GcatWiki - User contributions [en]

IGEM 2009 Project

2009-06-29T20:06:19Z

ShPunjabi:

Biology Based:
#[[Which reporters are we going to use?]]
#[[What naming system are we going to use for the suppressor tRNAs?]]
#[[How do you build the tRNA construct?]]
#[[How are we going to build the 5-mer BioBricks?]]

[http://gcat.davidson.edu/GcatWiki/images/2/2c/Stop_Codons_in_LCs.doc Using Stop Codons to Truncate Translation] 
[http://gcat.davidson.edu/GcatWiki/images/8/81/D5merSequences.doc Davidson ATG+5mer BioBrick Sequences] 
[http://gcat.davidson.edu/GcatWiki/images/4/47/FML_CODING_SEQUENCE_2.doc Frameshift Mutation Leader PCR primers] 
[http://www.staff.uni-bayreuth.de/~btc914/search/index.html Align E. coli tRNA sequences] 
[http://media.pearsoncmg.com/bc/bc_campbell_genomics_2/medialib/jmol/trna/index.html Compare 2 tRNA structures] 

'''Team Progress Table'''

{| border="1" cellpadding="2"
!width="120"|Student Name
!width="30"|5mer Codon
!width="40"|Anticodon Sequence
!width="40"| [http://gcat.davidson.edu/GcatWiki/index.php/What_naming_system_are_we_going_to_use_for_the_suppressor_tRNAs%3F Codon Short Name]
!width="150"|tRNA Status
!width="150"|5mer+XFP Status
!width="150"|5mer+Drug resistance Status
|-
| Olivia Ho-Shing || CCAUC || GUGAUCCAA-9 || Pro4 || 2 clones being resequenced || 2 5mer+RFPs sequencing || 5 5mer+Tets sequencing
|-
| Olivia Ho-Shing || CCAUC || UUUGAUGGAG-10 || Pro5 || 1 clone being resequenced || 2 5mer+RFPs sequencing || 5 5mer+Tets sequencing
|-
| Romina Clemente|| CUAGU || UUACUAGAC-9 || Leu4|| '''1 clone 100% match''', putting pBAD upstream || 9 5mer+RFPs sequencing || 5 5mer+Tets sequencing
|-
| Shamita Punjabi|| CCACU || CUAGUGGAC-9 || Pro3|| 2 clones being resequenced || 6 5mer+RFPs sequencing || 5 5mer+Tets sequencing
|-
| Leland Taylor || CCCUC || CUGAGGGUC-9 || Pro6 || '''1 clone 100% match''', putting pBAD upstream || 7 5mer+RFPs sequencing || 7 5mer+Tets sequencing
|-
| Alyndria Thompson|| CGGUC || UUGACCGAC-9 || Arg2 || 5 clones being sequenced || 4 5mer+RFPs sequencing || 6 5mer+Tets sequencing
|-
| Clif Davis|| 5mer codon || Anticodon Sequence || Codon Short Name|| tRNA Status || 5mer+RFP Status || 2 5mer+CAT sequencing
|-
| Student Name|| 5mer codon || Anticodon Sequence || Codon Short Name|| tRNA Status || 5mer+RFP Status || 5mer+Tet Resistance Status
|-
| Student Name|| 5mer codon || Anticodon Sequence || Codon Short Name|| tRNA Status || 5mer+RFP Status || 5mer+Tet Resistance Status
|-
| Student Name|| 5mer codon || Anticodon Sequence || Codon Short Name|| tRNA Status || 5mer+RFP Status || 5mer+Tet Resistance Status
|-
| Student Name|| 5mer codon || Anticodon Sequence || Codon Short Name|| tRNA Status || 5mer+RFP Status || 5mer+Tet Resistance Status
|-
| Student Name|| 5mer codon || Anticodon Sequence || Codon Short Name|| tRNA Status || 5mer+RFP Status || 5mer+Tet Resistance Status
|-
|}

Math Based: (Davidson) We have posted a Mathematica notebook on the Wiggio. Basically we are at a point where we can choose the number of variables, SAT number(2-SAT, 3-SAT, etc), and the number of clauses to use at a time and produce a table of inputs as the rows and clauses as the columns, with a 1 if the input satisfies the clause and a 0 if the input fails to satisfy the clause. In addition, we can produce a "Super Table," which lists all the SAT problems given the number of clauses in conjunctive normal form and displays how many of the clauses in a given problem each input satisfies. Feel free to view the posted Mathematica notebook; there are some instructions within the notebook itself. When you open it, there will be a window which asks about dynamic content; be sure to click "Enable Dynamic."

IGEM 2009 Project

2009-06-29T20:05:15Z

ShPunjabi:

Biology Based:
#[[Which reporters are we going to use?]]
#[[What naming system are we going to use for the suppressor tRNAs?]]
#[[How do you build the tRNA construct?]]
#[[How are we going to build the 5-mer BioBricks?]]

[http://gcat.davidson.edu/GcatWiki/images/2/2c/Stop_Codons_in_LCs.doc Using Stop Codons to Truncate Translation] 
[http://gcat.davidson.edu/GcatWiki/images/8/81/D5merSequences.doc Davidson ATG+5mer BioBrick Sequences] 
[http://gcat.davidson.edu/GcatWiki/images/4/47/FML_CODING_SEQUENCE_2.doc Frameshift Mutation Leader PCR primers] 
[http://www.staff.uni-bayreuth.de/~btc914/search/index.html Align E. coli tRNA sequences] 
[http://media.pearsoncmg.com/bc/bc_campbell_genomics_2/medialib/jmol/trna/index.html Compare 2 tRNA structures] 

'''Team Progress Table'''

{| border="1" cellpadding="2"
!width="120"|Student Name
!width="30"|5mer Codon
!width="40"|Anticodon Sequence
!width="40"| [http://gcat.davidson.edu/GcatWiki/index.php/What_naming_system_are_we_going_to_use_for_the_suppressor_tRNAs%3F Codon Short Name]
!width="150"|tRNA Status
!width="150"|5mer+XFP Status
!width="150"|5mer+Drug resistance Status
|-
| Olivia Ho-Shing || CCAUC || GUGAUCCAA-9 || Pro4 || 2 clones being resequenced || 2 5mer+RFPs sequencing || 5 5mer+Tets sequencing
|-
| Olivia Ho-Shing || CCAUC || UUUGAUGGAG-10 || Pro5 || 1 clone being resequenced || 2 5mer+RFPs sequencing || 5 5mer+Tets sequencing
|-
| Romina Clemente|| CUAGU || UUACUAGAC-9 || Leu4|| '''1 clone 100% match''', putting pBAD upstream || 9 5mer+RFPs sequencing || 5 5mer+Tets sequencing
|-
| Shamita Punjabi|| CCACU || CUAGUGGAC-9 || Pro3|| Running and gel purifying 2 tRNAs for ligation || 6 5mer+RFPs sequencing || 5 5mer+Tets sequencing
|-
| Leland Taylor || CCCUC || CUGAGGGUC-9 || Pro6 || '''1 clone 100% match''', putting pBAD upstream || 7 5mer+RFPs sequencing || 7 5mer+Tets sequencing
|-
| Alyndria Thompson|| CGGUC || UUGACCGAC-9 || Arg2 || 5 clones being sequenced || 4 5mer+RFPs sequencing || 6 5mer+Tets sequencing
|-
| Clif Davis|| 5mer codon || Anticodon Sequence || Codon Short Name|| tRNA Status || 5mer+RFP Status || 2 5mer+CAT sequencing
|-
| Student Name|| 5mer codon || Anticodon Sequence || Codon Short Name|| tRNA Status || 5mer+RFP Status || 5mer+Tet Resistance Status
|-
| Student Name|| 5mer codon || Anticodon Sequence || Codon Short Name|| tRNA Status || 5mer+RFP Status || 5mer+Tet Resistance Status
|-
| Student Name|| 5mer codon || Anticodon Sequence || Codon Short Name|| tRNA Status || 5mer+RFP Status || 5mer+Tet Resistance Status
|-
| Student Name|| 5mer codon || Anticodon Sequence || Codon Short Name|| tRNA Status || 5mer+RFP Status || 5mer+Tet Resistance Status
|-
| Student Name|| 5mer codon || Anticodon Sequence || Codon Short Name|| tRNA Status || 5mer+RFP Status || 5mer+Tet Resistance Status
|-
|}

Math Based: (Davidson) We have posted a Mathematica notebook on the Wiggio. Basically we are at a point where we can choose the number of variables, SAT number(2-SAT, 3-SAT, etc), and the number of clauses to use at a time and produce a table of inputs as the rows and clauses as the columns, with a 1 if the input satisfies the clause and a 0 if the input fails to satisfy the clause. In addition, we can produce a "Super Table," which lists all the SAT problems given the number of clauses in conjunctive normal form and displays how many of the clauses in a given problem each input satisfies. Feel free to view the posted Mathematica notebook; there are some instructions within the notebook itself. When you open it, there will be a window which asks about dynamic content; be sure to click "Enable Dynamic."

IGEM 2009 Project

2009-06-26T15:35:41Z

ShPunjabi:

Biology Based:
#[[Which reporters are we going to use?]]
#[[What naming system are we going to use for the suppressor tRNAs?]]
#[[How do you build the tRNA construct?]]
#[[How are we going to build the 5-mer BioBricks?]]

[http://gcat.davidson.edu/GcatWiki/images/2/2c/Stop_Codons_in_LCs.doc Using Stop Codons to Truncate Translation] 
[http://gcat.davidson.edu/GcatWiki/images/8/81/D5merSequences.doc Davidson ATG+5mer BioBrick Sequences] 
[http://gcat.davidson.edu/GcatWiki/images/4/47/FML_CODING_SEQUENCE_2.doc Frameshift Mutation Leader PCR primers] 
[http://www.staff.uni-bayreuth.de/~btc914/search/index.html Align E. coli tRNA sequences] 
[http://media.pearsoncmg.com/bc/bc_campbell_genomics_2/medialib/jmol/trna/index.html Compare 2 tRNA structures] 

'''Team Progress Table'''

{| border="1" cellpadding="2"
!width="200"|Student Name
!width="30"|5mer Codon
!width="40"|Anticodon Sequence
!width="40"| [http://gcat.davidson.edu/GcatWiki/index.php/What_naming_system_are_we_going_to_use_for_the_suppressor_tRNAs%3F Codon Short Name]
!width="150"|tRNA Status
!width="150"|5mer+RFP Status
!width="150"|5mer+Tet Resistance Status
|-
| Olivia Ho-Shing || CCAUC || GUGAUCCAA-9 || Pro4 || 4 clones being sequenced || Amplifying 5mer+RFP || Amplifying 5mer+Tet
|-
| Olivia Ho-Shing || CCAUC || UUUGAUGGAG-10 || Pro5 || 3 clones being sequenced || Amplifying 5mer+RFP || Amplifying 5mer+Tet
|-
| Romina Clemente|| CUAGU || UUACUAGAC-9 || Leu4|| 1 clone being sequenced || Gel purifying 5mer+RFP || Gel purifying 5mer+Tet Resistance
|-
| Shamita Punjabi|| CCACU || CUAGUGGAC-9 || Pro3|| 2 clones being re-sequenced || 6 RFP clones being sequenced || 5 Tet clones being sequenced
|-
| Leland Taylor || CCCUC || CUGAGGGUC-9 || Pro6 || 2 clones being sequenced || Transformed 5mer+RFP || Transformed 5mer+Tet
|-
| Alyndria Thompson|| CGGUC || UUGACCGAC-9 || Arg2 || 5 clones being sequenced || Transformed || Transformed
|-
| Student Name|| 5mer codon || Anticodon Sequence || Codon Short Name|| tRNA Status || 5mer+RFP Status || 5mer+Tet Resistance Status
|-
| Student Name|| 5mer codon || Anticodon Sequence || Codon Short Name|| tRNA Status || 5mer+RFP Status || 5mer+Tet Resistance Status
|-
| Student Name|| 5mer codon || Anticodon Sequence || Codon Short Name|| tRNA Status || 5mer+RFP Status || 5mer+Tet Resistance Status
|-
| Student Name|| 5mer codon || Anticodon Sequence || Codon Short Name|| tRNA Status || 5mer+RFP Status || 5mer+Tet Resistance Status
|-
| Student Name|| 5mer codon || Anticodon Sequence || Codon Short Name|| tRNA Status || 5mer+RFP Status || 5mer+Tet Resistance Status
|-
| Student Name|| 5mer codon || Anticodon Sequence || Codon Short Name|| tRNA Status || 5mer+RFP Status || 5mer+Tet Resistance Status
|-
|}

Math Based: (Davidson) We have posted a Mathematica notebook on the Wiggio. Basically we are at a point where we can choose the number of variables, SAT number(2-SAT, 3-SAT, etc), and the number of clauses to use at a time and produce a table of inputs as the rows and clauses as the columns, with a 1 if the input satisfies the clause and a 0 if the input fails to satisfy the clause. In addition, we can produce a "Super Table," which lists all the SAT problems given the number of clauses in conjunctive normal form and displays how many of the clauses in a given problem each input satisfies. Feel free to view the posted Mathematica notebook; there are some instructions within the notebook itself. When you open it, there will be a window which asks about dynamic content; be sure to click "Enable Dynamic."

IGEM 2009 Project

2009-06-19T20:53:10Z

ShPunjabi:

Biology Based:
#[[Which reporters are we going to use?]]
#[[What naming system are we going to use for the suppressor tRNAs?]]
#[[How do you build the tRNA construct?]]
#[[How are we going to build the 5-mer BioBricks?]]

[http://gcat.davidson.edu/GcatWiki/images/2/2c/Stop_Codons_in_LCs.doc Using Stop Codons to Truncate Translation] 
[http://gcat.davidson.edu/GcatWiki/images/8/81/D5merSequences.doc Davidson ATG+5mer BioBrick Sequences]

[http://gcat.davidson.edu/GcatWiki/images/4/47/FML_CODING_SEQUENCE_2.doc Frameshift Mutation Leader PCR primers]

'''Team Progress Table'''

{| border="1" cellpadding="2"
!width="200"|Student Name
!width="30"|5mer Codon
!width="40"|Anticodon Sequence
!width="40"|Codon Short Name
!width="150"|tRNA Status
!width="150"|5mer+RFP Status
!width="150"|5mer+Tet Resistance Status
|-
| Olivia Ho-Shing || CCAUC || GUGAUCCAA-9 || Pro4 || 4 clones being sequenced || Amplifying 5mer+RFP || Amplifying 5mer+Tet
|-
| Olivia Ho-Shing || CCAUC || UUUGAUGGAG-10 || Pro5 || 3 clones being sequenced || Amplifying 5mer+RFP || Amplifying 5mer+Tet
|-
| Romina Clemente|| CUAGU || UUACUAGAC-9 || Leu4|| 1 clone being sequenced || Gel purifying 5mer+RFP || Gel purifying 5mer+Tet Resistance
|-
| Shamita Punjabi|| CCACU || CUAGUGGAC-9 || Pro3|| 3 clones being sequenced || Cleaning/Concentrating 5mer+ RFP || Cleaning+Conc 5mer +Tet
|-
| Student Name|| 5mer codon || Anticodon Sequence || Codon Short Name|| tRNA Status || 5mer+RFP Status || 5mer+Tet Resistance Status
|-
| Student Name|| 5mer codon || Anticodon Sequence || Codon Short Name|| tRNA Status || 5mer+RFP Status || 5mer+Tet Resistance Status
|-
| Student Name|| 5mer codon || Anticodon Sequence || Codon Short Name|| tRNA Status || 5mer+RFP Status || 5mer+Tet Resistance Status
|-
| Student Name|| 5mer codon || Anticodon Sequence || Codon Short Name|| tRNA Status || 5mer+RFP Status || 5mer+Tet Resistance Status
|-
| Student Name|| 5mer codon || Anticodon Sequence || Codon Short Name|| tRNA Status || 5mer+RFP Status || 5mer+Tet Resistance Status
|-
| Student Name|| 5mer codon || Anticodon Sequence || Codon Short Name|| tRNA Status || 5mer+RFP Status || 5mer+Tet Resistance Status
|-
| Student Name|| 5mer codon || Anticodon Sequence || Codon Short Name|| tRNA Status || 5mer+RFP Status || 5mer+Tet Resistance Status
|-
| Student Name|| 5mer codon || Anticodon Sequence || Codon Short Name|| tRNA Status || 5mer+RFP Status || 5mer+Tet Resistance Status
|-
|}

Math Based: (Davidson) We have posted a Mathematica notebook on the Wiggio. Basically we are at a point where we can choose the number of variables, SAT number(2-SAT, 3-SAT, etc), and the number of clauses to use at a time and produce a table of inputs as the rows and clauses as the columns, with a 1 if the input satisfies the clause and a 0 if the input fails to satisfy the clause. In addition, we can produce a "Super Table," which lists all the SAT problems given the number of clauses in conjunctive normal form and displays how many of the clauses in a given problem each input satisfies. Feel free to view the posted Mathematica notebook; there are some instructions within the notebook itself. When you open it, there will be a window which asks about dynamic content; be sure to click "Enable Dynamic."

IGEM 2009 notebook

2009-06-18T20:49:27Z

ShPunjabi: /* Roclemente 14:00, 18 June 2009 (EDT) */

== [[User:Roclemente|Roclemente]] 16:54, 3 June 2009 (EDT) ==

Shamita and I are trying to find suitable reporter proteins to use. Yesterday, Leland and Alyndria were working on ways to insert the gene sequences into the plasmid. Upon seeing how they wanted to manipulated the reporter gene to include the logical clauses, we came up with a few criteria for the reporter genes we would use. The following criteria for genes are listed in order of the broadest aspect to look at to the narrowest aspect:

a) Doesn't contain restriction sites for the 4 restriction enzymes (EcoR1, Xbal, Spel, Pst1) used to cleave the Biobrick part out of the plasmid.

b) Contains 6 cutter restriction sites.

c) These restriction enzymes aren't blunt (cleave straight down at one spot).

d) These restriction sites are close to thge 5' (beginning) end of the sequence.

e) These enzymes are easiest to work with and cheapest.

We are finding the part numbers of the reporter genes we want to use (antibiotic resistance, fluorescence, ''LacZ'') through our own GCAT because we know these ones work. We are then locating these parts on the parts registry [http://www.partsregistry.org] website. We copied and pasted the gene sequences we obtained from the registry onto the ApE software [http://www.biology.utah.edu/jorgensen/wayned/ape/]. From here, we were able to generate a genetic map of each gene that outlined each restriction site that fit our criteria. We put each genetic map, alongside the part number used, into a Word document:

http://gcat.davidson.edu/GcatWiki/images/0/0e/Restriction_Site_Mapping_on_Reporter_Genes.doc

So it looks like we've changed directions. The team is looking towards the wet lab portion of our research more now. Instead of simply speculating about which reporter proteins we think will work best and which tRNA suppressors to use, we're going to physically test it out ourselves. A few tasks at hand in the second part of this afternoon:

1. What are the DNA gene sequences of the 12 different tRNA suppressors we want to use?

2. What is the sequence of the DNA that is cleaved off of both ends of the tRNA when it is transcribed?

We e-mailed Anderson to find out the exact tRNA gene sequences because we had a hard time finding this information everywhere else. Most papers we found describing the 5-base codon tRNA suppressors simply referred to Anderson's paper [http://gcat.davidson.edu/GcatWiki/images/6/6f/AndersonSchultz2002ChemBiol.pdf]. Anderson replied near the end of the day with the gene sequence. According to his email, the only difference between different tRNA genes is the anticodon loop. Therefore, if we just substitute all the anticodon loops in the invariable part of the sequence, we have our different tRNA sequences. We plan on using the Lancilator to help us break these tRNA sequences into smaller oligonucleotides that can anneal at around the same temperature because it is not possible for us to order oligos that are greater than 70 nucleotides long.

== [[User:Roclemente|Roclemente]] 17:21, 4 June 2009 (EDT) ==

I started out the day looking at my time-sensitive group research qustion about reporter proteins. I started making a table listing the advantages and disadvantages of different reporter proteins. Then the group found out that there was a major roadblock to our project: a stop codon was located in our ATG-5mer oligo. The stop codon would have been part of the BioBrick scar. The group looked at several ways to work around this problem. The idea that I looked most into was replacing the restriction sites that were used in the scar through standard assembly. According to the judging criteria for iGEM, I found that a team could possibly alter the standard assembly method as long as they wrote a letter to the iGEM judges explaining our plan in a detailed manner. From there, I set off to understand the BioBrick scar more by making a document highlighting the exact positions of the rsetriction sites on the BioBrick parts and how they were used to put more than one part together. I then found several other pairs of restriction sites that complemented one another and could be used in place of the standard scar between Xba1 and Spe1. At the end of the day, the group got the chance to speak with Dr. Campbell and he suggested a hybrid idea which would combine Davidson's restriction site idea with Missouri's PCR idea. I'm not quite sure of what this idea is exactly yet, but we'll all clarify our vague idea of it tomorrow morning after a talk with Dr. Eckdahl.

== [[User:Roclemente|Roclemente]] 17:13, 5 June 2009 (EDT) ==

This morning we began exploring different options for bypassing the stop codon problem from yesterday. If we wanted to make sure we were using the BioBrick parts in our GCatalog, there was no way we were going to be getting rid of the Xba1 restriction site that contained the "TAG" which codes for a stop codon. Therefore, we proposed looking at restriction sites within the reporter genes instead, therefore addressing the already-placed BioBrick ends. Using the document me and Shamita created on "Restriction Site Mapping on Reporter Genes" (see first journal entry), we determined that the reporter gene with the restriction site closest to the beginning of the sequence was YFP. We proposed cutting the YFP gene that we already had in stock at this restriction site and inserting an oligo (which we would have ordered) with a sequence in the following order: "BioBrick prefix - ATG - 5mer - part of YFP gene to the left of the cleaved restriction site."

After clarifying our own idea with one another, we spoke with some Missouri kids to clarify the said "hybrid" idea which, according to Dr. Campbell, combined the ideas of both campuses. The hybrid idea was based upon our idea of cutting the reporter gene at a restriction site as well, except that our primer would start out with a primer for our reporter gene, and to the left of the primer would be a "tail" consisting of the ATG and 5mer and either an Xba1 or EcoR1 restriction site (We still have to decide this. Our decision will probably be based on the restriction site we end up using in our reporter gene and whether or not that is complementary to either the Xba1 or EcoR1 sites.). We would use the PCR technique and use our in-stock reporter gene strand as our template strand (it will be denatured into single strands) and we will add our oligo. We propose that the primer will synthesize the rest of the YFP strand as well as add on nucleotides to complete the oligo tail. In the end, we will have a double stranded DNA sequence consisting of either the Xba1 or EcoR1 site, the ATG, the 5mer, and the reporter gene. The beauty of this hybrid idea is that we have flexibility as far as the reporter gene we choose to use since all we really need for it is the beginning sequence (for the primer) and a known, "hearty" restriction site. Once we cut this double strand at this restriction site on the reporter gene as well as at the Xba1 or EcoR1 sites, we can insert this into a plasmid that contains the rest of that reporter gene after the restriction site we choose to use and the complementary Xba1 or EcoR1 site.

I made a Word document outlining the promoter and RBS parts we have in our registry on the Davidson campus. Leland and I went through the registry parts on the GCATalog to look for potential promoters and RBS parts. We went through the document as a group and analyzed each of these potential parts on partsregistry.org to see which ones would most likely work the best in the miniprep we'll start preparing for on Sunday evening. Here is the document we wound up with:

http://gcat.davidson.edu/GcatWiki/images/d/d2/Promoters_and_RBS.doc

== [[User:Roclemente|Roclemente]] 10:40, 8 June 2009 (EDT) ==

We're starting the day by finalizing the oligos we will be ordering to have synthesized. Two groups of oligos must be sequenced: primers and tRNA gene sequence. The primers will consist of a forward and reverse primer. The forward primer will consist of the following in this 5' - 3' order: GCAT - BioBrick Prefix - ATF - 5mer - first 20 nucleotides in the reporter gene sequence. These 20 nucleotides won't include the ATG that naturally comes at the beginning of the reporter gene because we want to make sure the ribosome attaches to the first ATG and reads the logical clause instead of going straight to the ATG at the start of the reporter gene. The reverse primer will consist of the first 20 nucleotides downstream of the restriction site we plan to use in the reporter gene. I worked on finding the primers for the Tet resistance gene (part J31007) while Lyn worked on finding the primers for the RGP gene. Dr. Campbell decided we should use the BamH1 restriction enzyme because it is very "hardy." The following document outlines the 5 forward primers (each with one of the five variable 5mers) we will order for amplification of the Tet resistance FML:

[[Image:Tet resistance forward primers.png]]

This is the reverse primer we will order for the Tet resistance FML:

[[Image:Tet resistsance reverse primer.png]]

== [[User:Roclemente|Roclemente]] 11:54, 9 June 2009 (EDT) ==

We need to digest the plasmids at the EcoR1 and Pst siets. Everytime we're going to set up a digestion, we'll want the following in these volumes: 10x Buffer (2 uL), DNA (~ 3 uL), enzymes 1 and 2 (1 uL each), water (13 uL), and final volume of all of these put together should be 20 uL.

Some notes about digestion: Enzymes should make up 10% of the final volume. As a general rule of thumb, make sure the tip of your pipet is close to the surface of the solution. This is especially pertinent when gathering 1 microL of each of the enzymes. The most sensitive components are the enzymes, so they should go last.

We'll have a bunch of tubes with each 3 microL of the specific DNA. We'll make a cocktail in a bigger tube consisting of the buffer, 2 enzymes, and water. The rationale behind thissis that you'll be less likely to make a bigger mistake with a bigger volume (versus getting 1 microL of enzyme per specific DNA). You'll also know that there is no problem with the enzyme if all the DNA don't cut. If only one specific DNA doesn't work, then we'll know that it's because there's something wrong with the specific DNA rather than anything in the "Master Mix." Since we have to do 11 digestions, we'll make enough of the master mix for 12 tubes to make sure we have enough. We'll take 17 microL of the Master Mix to each tube of DNA.

The optimal temperature for most enzymes is 37 degreees celsius. You'd like to have enzymes with the same optimal temperatures (for ex. restriction enzymes that start with B usually have an optimal temperature near 50 degrees. You wouldn't want to use both B-enzyme and EcoR1 because EcoR1 would die).

'''What percent gel should we use?'''

Look at the Davidson lab protocol on the Davidson-Missouri Wiki. Generally, bigger molecules require an agarose gel with a lower concentration.

'''What's next in the future?'''

5' end additions are done by PCR. We'll ampliffy this double-stranded DNA. We'll cut with both EcoR1 and BamH1 or Nco1 (depending on which reporter gene you're using). Then we'll cut the plasmid with the reporter with the same restriction enzymes used.Then, to throw away the remaining DNA that we don't want, we'll run a gel and throw away the smaller band (because the larger band is probably made of the plasmid we want to keep).Then we'll take the double stranded DNA we amplified and the purified plasmid and ligate, transform, and screen them and verify the sequence.

While we wait for the oligos, we can cut the plasmid with reporter on the same restriction enzyme already.

'''How are we building the tRNAs?'''

We'll assemble the oligos into genes by mixing our oligos together, boiling them, and very slowly allowing them to cool. This will require many calculations. We'll cut the plasmids with EcoR1 and Pst1. to be ready to receive the enes we're assembling.We'll have to gel purify the plasmids to get them away from their inserts. We'll ligate, transform, and screen the genes and the plasmids and verify the sequence. On paper you shouldn't have two sites cut with different restriction enzymes stick together, but if there's selection pressure, these sites may cut some ends to fit together. This is why we must screen them.

After Dr. Campbell taught us all of the above, the 5 biologists put together a document outlining exactly how we would go about cutting the two sets of plasmids (one set will be cut with Eco R1 and Pst1 and othe other set will be cut with Eco R1 and BamH1 or Nco1). The following table outlines which biologist was going to work with which sample of DNA:

[[Image:Names and DNA.jpg]]

The following summarize the diferent materials and volumes of these materials that we'll need for our digestion of the plasmids:

[[Image:Digestion materials.png]]

This last table highlights the agarose percent concentration we should use for each part according to how many base pairs it contains:

[[Image:Screen-capture-3.png]]

The biologists digested the plasmids that we plan on using once our oligos arrive in a day or two. Afterwards, we made the two agarose gels that were most compatible with the parts we were going to run. This gel is going to purify the plasmids so that we have the parts that we're going to use later on, minus the rest of the DNA in the plasmid that we don't need. Before physically creating the gel, we figured out how much agarose we would need to make our two different agarose gel concentrations, 0.8% and 2.5%. Next, we made sure the mold and the big teeth were in place. We used the big teeth at the end of the mold because we plan on running 20 uL of DNA versus a running a smaller portion which would require us to use small teeth. We then measured the amount of agarose that we calculated we would need, placed this in a volumetric flask greater than 60 uL to allow for fizz to rise up, then measured out 60 uL of buffer and mixed this in the same flask with the agarose. We placed clear plastic wrap on top of the flask and heated it up for 1 minute and 20 seconds in the microwave. After we heated it, we took it out and checked to see if there was still agarose present that hadn't yet melted. There was, so we placed it back in the microwave and heated it for another 20 seconds. All the agarose had melted. We added 1 uL of EtBr (ethidium bromide) to the solution and quickly poured this into the mold. It needs about 15 to 20 minutes to cool. Dr. Campbell made the agarose gel that was 2.5% concentrated gel while we watched. We then made the 0.8% concentrated gel while he watched.

The following table lists the order in which we loaded our DNA into the gel. The first column all the way to the left is represented by number 1:

[[Image:Screen-capture-2.png]]

== [[User:Roclemente|Roclemente]] 08:46, 10 June 2009 (EDT) ==

After about 30 minutes after we began the gel electrophoresis, we looked at our gels with UV light to see how far the inserts had travelled and where the plasmids were. Then we took each gel to be photographed in the freezer room (231). We used the following DNA length marker to measure how long each sequence represented by each band was:

[[Image:Screen-capture-1.png]]

We compared our data to the lengths we expected to get according to the registry online. The first gel we photographed was the one that was 2.5% concentrated:

[[Image:2009-06-09 14hr 44min.jpg]]

The first two columns of the DNA we ran, both consisting of B0300, were fairly the same as the length we expected. However, the last two columns, consisting of K091111 and K091112 respectively were supposed to be 86 bp long, however, according to our data, K091112 was longer. Leland and I looked at Pallavi Penumetcha's paper which delved more closely into these two parts and what she wrote in her paper and what she entered into the registry conflict. We are currently corresponding with her to figure out the exact discrepancy.

We then photographed the 0.8% gel:

[[Image:2009-06-09 15hr 21min.jpg]]

The length of the sequences in almost every column matched up with the lengths we expected. Only the 4th and 5th columns didn't come out clearly. We think that the DNA inserted in column 5 weren't inserted properly and diffused on its own. We couldn't see bands in the 4th column at all until we adjusted the color a bit on the photographer and saw that column 4 was pretty much identical to column 5. We assumed that column 5 bled into column 4.

== [[User:Roclemente|Roclemente]] 15:14, 11 June 2009 (EDT) ==

We are digesting two parts today: I715039 (pLac+RBS+RFP) and S03710 (Tet Resistance). We want both the plasmids and the inserts in each case. We are aiming for a final volume of 40 uL for each part. This means that each digestion will consist of 4 uL buffer, 2 uL EcoR1, and 2 uL of their other respective restriction site (BamH1 for S03710 and Nco1 for I715039). The only parts that will vary are the amount of DNA and water we use. The amount of DNA we use depends on the concentration of the DNA we have (found through use of the NanoDrop). Since we want 2500 ng of DNA, we can use the concentration of DNA to figure out how much volume of this concentrated DNA we'll want to use in your digestion. If we subtract the volumes of buffer, two restriction enzymes, and DNA we plan on using from 40 uL, we'll have the amount of water we need to add. Here are the different volumes of materials we plan on using in our double digestion:

[[Image:DoubledigestRFPTetR.png]]

Now that we know how much of each material we were going to use, we needed to find out which buffer would be compatible with each sample according to the restriction enzymes we were using and how effective they'd be in different salt solutions. Online on the Promega "Compatible Buffers" website, we were able to find which Promega buffers would be most compatible when doing a double digest with EcoR1 and BamH1:

[[Image:BamEcoR1.png]]

Here is a chart noting buffers and their compatiblity with EcoR1 and Nco1:

[[Image:NcoEcoR1.png]]

We still have to wait for the restriction enzymes to arrive, so afterwards, we transformed three different inserts containing needed promoters and RBSs into vectors. We used the Zippy Transformation method. These promoters and RBSs will later be added to the FMLs once they are already inserted into the reporter cells. The following table outlines these inserts and part numbers:

[[Image:Promoterinsertspartnumbers.png]]

The restriction enzymes have arrived. We will now be able to do PCR to amplify the RFP gene (I715039) and the Tet resistance gene (S03710) as templates. We will be using the primers which have also arrived. We ordered forward and reverse primers so that our amplified sequence will consist of the following in this order:

GCAT-ATG-5mer-Reporter Gene

The following table lists the materials will be needed for this PCR technique which uses the "Green Promega master (Monster) Mix:"

[[Image:Greenpromegamastermix.png]]

This is the PCR procedure:

[[Image:Temperaturecycle.png]]

These are the variables that we will be adding to each separate sequence we want to be amplified:

[[Image:Tobeamplified.png]]

This table shows how we labeled the tubes based on how we divided the PCR work for the 10 tubes:

[[Image:Pcrtubes.png]]

Now we are giong to assemble the tRNA oligos together to form the tRNA genes. We could only order a certain length of sequence and the tRNA genes outdid these specified lengths. We used the Lancilator to find various oligonucleotides that overlapped and would be able to anneal together to form the tRNA genes. We used the Davidson lab protocol entitled "Buiding dsDNA with Oligos" to assemble the oligos:

[[Image:Oligoassembly.png]]

== [[User:Roclemente|Roclemente]] 22:46, 12 June 2009 (EDT) ==

We had two main goals for today:
1. perform a tRNA ligation and transformation
2. perform two electrophoreses: one to verify the PCR sequences we obtained and another to purify the digested reporter plasmids we would need

We began the day by pouring the agarose gels according to which concentration of buffer would be most compatible with the molecular weight of the substance we wanted to isolate. For both the digestion of E01010 and J31007, according to the "Optimize your Gel" website found on the Davidson Lab Protocols site for iGEM 2009, we wanted a 0.4% concentrated gel. For the PCRs, we wanted a 1.6% concentrated gel to optimize our retrieval of the Tet gene and a 1.4% concentrated gel to optimize our retrieval of the RFP gene.

While we waited for the gels to harden for about 30 minutes, we began calculating how much of each material we would need to ligate our tRNAs. We used the following equation to find out how much insert we should use:

[[Image:Ligationequation.png]]

Using the lengths of sequences we would be ligating, this is the mass that we needed:

X nanograms of insert = [(2)(144bp)(50 ng linearalized plasmid)]/2038bp
X = 7.0657 ng of insert

The following table outlines our calculations for finding the mass of tRNAs we had:

[[Image:Ligationcalculations.png]]

We would attain this mass of tRNA by pipetting an unknown amount of it. In order to attain a more "pipetable" amount of tRNA, we diluted our initial concentration 50X. Since we knew we wanted our final mass to be 7.065 ng, we calculated the volume we would need to take from this new, diluted sample:

DILUTION: 555.48/50 = 11.012
CALCULATION: 11.012 ng/ 1uL = 7.065 ng/ Y uL
Y = .64 uL of insert

Afterwards, Olivia and Alyndria began the actual ligation process while Shamita, Leland, and I filled the wells of the two gels. The first included the two digested reporter parts. We also included a molecular weight marker. Here is a photograph of this gel:

[[Image:2009-06-12 15hr 54min.jpg]]

According to our prior calculations, the Tet plasmid should have been 2958 base pairs long and the RFP plasmid should have been 2380 base pairs long. Our expectations were verified by the data. Shamita and I then sliced the part of thsi gel containing both plasmids and put these slices away to purfiy on Monday.

The second gel contained aliquots of the PCR we had done to attain 10 FML sequences: 5 containing the Tet resistant reporter gene and each of the 5 frameshifts that Davidson was assigned and the other 5 containing the RFP reporter gene and each of the 5 frameshifts as well:

[[Image:2009-06-12 16hr 07min.jpg]]

According to our data, each sequence containing the Tet resistant gene should have been 317 base pairs long and each sequence containing the RFP gene should have been 446 base pairs long. Our data supports our hypothesis, however, the 3rd well was not successful. This well contained the FML consisting of the Tet resistant gene and the frameshift mutant CUAGU. Before leaving work for the day, Shamita and I prepared and performed PCR using two parts containing the tet resistant gene, J31007 and S03710. We wanted to use both to make sure at least one of them works since we could technically use either of them, even though when we first did PCR we only used the S03710 part. Using the known concentrations of each of these parts, and knowing that we needed 1 ng of each part to include in our PCR samples, we calculated the volume of each 100X-diluted solution we would need:

S03710: 1.636 ng/uL = 1 ng/ X uL
X = 0.611 uL

J30017: 0.776 ng/uL = 1 ng/ Y uL
Y = 1.289 uL

We will run another gel with both of these parts on Monday and hopefully at least one of them works.

== [[User:Roclemente|Roclemente]] 17:24, 15 June 2009 (EDT) ==

Each biology member of the Davidson iGEM team has been assigned a 5mer to carry on throughout the rest of their respective processes. I've been assigned the 5mer CUAGU. We verified the PCR of the CUAGU FML containing the RFP gene on Friday. However, we weren't able to verify the CUAGU FML containing the tet resistance gene when we ran it on a gel. Shamita and I redid PCR for two parts containing the tet resistance gene: J31007 and S03710, labeled 2* and 2! respectively. I'm running another gel to verify either or both of them. Both sequences should be 317 bp long (this includes the ATF, logical clause, and the tet resistance gene to the left of the BamH1 restriction site). Here is the gel run we collected. The first column represents the 1 kb DNA marker, and the next two are 2* then 2!:

[[Image:2009-06-15 10hr 34min.jpg]]

The data shows that the PCRs of both parts wound up at the length we expected. The first time we did PCR on J31007, we must have left a solution out of our procedure on accident. I combined both PCRs together since they are the same length and match the length we expected to get.

Next, we cleaned and concentrated our amplified DNA sequence (#D4014 from Zymo Research). I cleaned the two pieces of DNA that contained the 5mer CUAGU. I labeled the tube with DNA also containing the RFP gene as "1" and the tube with DNA also containing the tet resistance gene as "2!". We needed 2 volumes of buffer for every 1 volume of DNA we added. Therefore, for tube "1," I added 95 uL DNA and 190 uL buffer. I added more DNA to tube "2!" since I combined the DNA from two PCR events to one (2* and 2! from earlier).

After cleaning the DNA, we found its concentration using the Nanodrop. In a solution made up of 0.5 uL of the DNA and 2.5 uL of TE, we found the following data from the DNA in this solution:

[[Image:Cleanconcentrate.png]]

The concentration of the DNA we cleaned was the concentration given by the Nanodrop multiplied by 6 (the DNA was dilute 6-fold for the Nanodrop). We used this concentration and the volume of clean DNA we knew we had (9.5 uL) to find the mass of clean DNA we had:

[[Image:Concentrateddna.png]]

Before doing digestion, we used the ligation equation (see "Ligation Protocol" in "Davidson Lab Protocols") to figure out how much mass of the DNA insert we would need later on when we did ligation. For the RFP FML, we found we would want the following mass of insert:

ng of insert = (2)(443)(50)/(2320) = 19.0948 ng

We multiplied this mass by 50 because we're counting on losing DNA between the digestion and cleaning we would need to do leading up to the ligation protocol.

19.0948 ng x 50 = 954.741 ng RFP FML

We found the mass of Tet FML insert we would need for ligation as well:

ng of insert = (2)(314)(50)/(2958) = 10.6153 ng

We multiplied this mass by 50 too:

10.6153 x 50 = 530.764 ng Tet FML

Unfortunately, nobody had enough of their clean DNA to satisfy our careful criteria. However, we would need to do this calculation later on so it was helpful we did it anyway. Instead, we just used all the DNA we had, or 9.5 uL for each reporter gene. Since there were 4 people who needed the same master mix, we made 5 parts of the master mix using the following volumes:

[[Image:Rfptetdigestion.png]]

After realizing that we would have to wait a day for the digestion to take place, we decided to culture every tRNA cell that colonized. The tRNA that we transformed on Friday did not have a good yield, so last night a few people came in and transformed more tRNA cells. We got a few more colonies. To be safe and make sure we cultured each tRNA, we picked every colony visible to us in both the old and new plates. Using our own given tRNAs, I counted the amount of colonies that grew in the plate we transformed on 6/12 and 6/14. There were 4 colonies that grew on both plates so I labeled a tube for each of these. I awas also in charge of the negative control. There were 2 colonies that grew on the negative control plate so I labeled 2 tubes for that. We will be placing them in an incubator overnight.

== [[User:Roclemente|Roclemente]] 12:30, 16 June 2009 (EDT) ==

We checked on the cells we cultured overnight. Of the cells that I cultured (all containing the CUAGU 5mer and the two negative controls), both negative control tubes worked as well as the tubes I labeled 1 (6/14), 5 (6/12), and 7 (6/12). The rest of mine were either red, meaning the plasmids hadn't ligated properly) or the colonies we used were no good. I took these 5 tubes, along with 3 of Olivia's tubes filled with cells that were cultured (tubes 11, 12, and 13) and miniprepped them to isolate the plasmids from the cells. There was no need to colony PCR to screen for successful ligations because this is only used to know which ones to let grow overnight and we had already done this.

As soon as I finished miniprepping them, I had to leave the lab for a little bit, so Olivia took each my miniprep tubes besides the negative controls and Leland took my negative controls. I completed the protocol for digestion of the tRNAs with EcoR1 and Pst1 for Alyndria's tubes when I got back because she had left a little earlier for lunch.

Afterwards, Shamita and I poured a 1.9% concentrated gel to load the digested tRNAs on. We want to verify that these tRNAs are the appropriate size for if they were cut properly. Since we will have to load 37 wells, and the maximum number we can load on one gel is 18 since 2 wells will have to be used by the molecular weight marker, we needed to pour 2 gels and we will also be using a half of a 2.0% agarose gel that was left over from another experiment.

Shamita and I also made new 0.5X running buffer gel. We did this by adding 450 uL buffer and 9 uL ethidium bromide (1 uL EtBr : 50 uL buffer). Alyndria, Leland, and Shamita loaded these gels.

Olivia and I cleaned our PCR DNA using the "Ethanol precipitate DNA (short protocol)" found in the Davidson Lab Protocols link. To begin with, I added 180 uL dH2O to my two samples, CUAGU with RFP and CUAGU with Tet, to bring their volumes up to 200 uL. During one of the last steps of this protocol, when Olivia and I dried out our DNA using the SpeedVac for a period of 10 minutes, the top of my tube containing the CUAGU 5mer and RFP gene broke. I wasn't sure if a pellet was in there, but I still poured out the ethanol and went on with the protocol. At the end, I resuspended what may or may not have been my DNA in 10 uL distilled water. I NanoDropped it afterward and attained the following data concerning both tubes:

[[Image:Failedpcrmeh.png]]

According to this data, any absorption and concentration we may have attained is insignificant because the A-260 must be between 0.05 and 1.50 to be significant and neither of the DNA is. Tomorrow I will do PCR for both FMLs over again.

== [[User:Roclemente|Roclemente]] 14:51, 17 June 2009 (EDT) ==

I found out that my tRNA DNA that I labeled "1" and which contains CUAGU was verified from the gel run Olivia did yesterday (the gel run also included her tRNA DNA samples). At the start of the day I plated this tube of cultured cells again to grow overnight.

Since PCR will take about 3 1/2 hours, I attempted this procedure at the beginning of the day. I plan on making 5 PCR products for each reporter gene since I now know the environmental conditions are correct. I labeled the RFP tubes 1-5 and the Tet tubes 6-10. I first needed to figure out how much of the template DNA I would need in order to have 1 ng template DNA. I made the following calculations to find this out:

[[Image:1ngtemplatedna.png]]

I wanted to add the Monster Mix last, so I made the following table, adding everything else besides the Monster Mix for 5 of the same reporter gene to one tube:

[[Image:Pcrmix.png]]

Unfortunately, when I actually mixed the solution together, I forgot to dilute the template DNA. Therefore, we have 100X more template DNA than we would have had we diluted the template DNA. The PCR can still work, but we will have to constantly remind ourselves that there will be extra template DNA in our solution and that this may skew, however slightly, our results later on.

While I waited for the PCR product, I refocused on getting my tRNA DNA ready for sequencing. Using the NanoDrop, I found the concentration of my miniprepped CUAGU DNA that was verified to be 61 ng/uL. The pre-sequencing protocol calls for 60 ng DNA, so I extracted 1 uL from this tube (the extra DNA will be insignificant in the sequencing procedure). I pipetted this into another tube and used the SpeedVac to dry it out. This is how we will send our DNA to be sequenced.

The following is a summary of steps I put together to help me plan out what I will do after the PCR procedure is over (it will be done at 5 pm today):

1. Combine the five matching tubes into one tube.
2. Run each PCR product on a gel to verify that our expected sequence has been properly amplified.
3. Clean and concentrate plasmids so salt solution is optimal for digestion.
4. Double digest PCR products with EcoR1 and BamH1 or Nco1.
5. Clean and concentrate again to get rid of restriction enzymes.
6. Nanodrop inserts in order to do ligation calculations and verify that sequences are present.
7. Ligate FML inserts with purified plasmids.
8. Transform plasmid into cells.

Since I won't have time to run a gel and take a picture once the PCR procedure is complete (we get out at 5:30 and it will be finished at 5:00), I have begun to prepare the agarose gel for use tomorrow. The size of the band I expect to find for the Tet FML is 349 bp (including the GCAT, BioBrick prefix, ATG, 5mer, and reporter gene until about 20 nucleotides or so past the restriction site we will cut at) and 474 bp for the RFP FML. According to the "Optimize Your Gel" link on the Davidson Lab Protocols page, a 1.6% gel will optimize how well I see my Tet bands and a 1.3% gel will optimize how well I see my RFP bands. I can use a gel with a concentration in between both of the optimal concentrations for Tet and RFP so that I can use one gel for both. Therefore, I will use a 1.45% gel. 1.45% of 60 uL buffer solution tells me that I should measure out 0.87 g agar for this gel.

== [[User:Roclemente|Roclemente]] 14:00, 18 June 2009 (EDT) ==

I checked on my PCR products this morning. Tubes 1 and 2 (both containing the RFP FML) had solution scattered on the tube's cover and walls so I placed them in the centrifuge for a bit. Unfortunately, I forgot to put these 500 uL tubes in holders when I centrifuged them so these tubes were destroyed. This was okay though because I was just going to combine each respective FML into one tube. I would have 300 uL of RFP FML PCR product instead of 500 uL and I put all of this in tube 5. I still had 500 uL of Tet FML PCR product left and put all of this in tube 10.

I ran 5 uL of both RFP and Tet FMLs on a 1.4% gel, as well as a 1 kb molecular weight marker. Since I suspected the RFP sequence to be 474 bp long, which would call for a 1.5% gel, and I also suspected the Tet sequence to be 349 bp long, I used a gel that was in between both concentrations so I could run both on one gel and get optimal results. The MW marker is in lane 1, the RFP is in lane 3, and the Tet is in lane 4 (Leland ran his PCR product in lane 2):

[[Image:2009-06-18 09hr 56min.jpg|thumb|none|500px|1.4% gel. Lane 1: MW marker. Lane 2: Leland's sequence. Lane 3: RFP PCR product. Lane 4: Tet PCR product.]]

I was expecting the RFP PCR product to be 474 bp long and the Tet PCR product to be 349 bp long. Both appear to come out a bit shorter, but Dr. Campbell and I agreed that we wouldn't count these sequences out because the bands were quite close to what we expected.

I proceeded to clean and concentrate these two sequences using the procedure outlined in the "Clean and Concentrate DNA (after PCR, before digestion)." Now I had 295 uL of the Tet sequence and 495 uL of the RFP sequence. Since the procedure says I can only have 800 uL in the column at a time, with DNA:Buffer at a ratio of 1:2, I mixed the two in a column in a series of two rounds using the following volumes:

[[Image:Clean.png]]

I Nanodropped my DNA after cleaning it and attained the following results:

[[Image:Nanome.png]]

I wanted the OD260 (Optical Density at 260) to be under 1.5, which it is for both. I lost quite a bit of Tet DNA in the process, and will only continue to lose DNA. To account for this loss of DNA, Dr. Campbell suggested it would be a good idea to heat inactivate the restriction enzymes to get rid of them after digestion instead of cleaning and concentrating the DNA over again.

I double digested both RFP and Tet FML sequences. I used all 9.5 uL of my DNA. The following table outlines how much volume of each material I mixed together:

[[Image:2nddoubledigest.png]]

While I waited 3 hours for my digestion to take place, I prepared 6 tubes to make more liquid cultures of the RFP and Tet vectors to grow more receiving vectors. 3 tubes would be to make RFP cultures and the other 3 would be for the Tet cultures. For RFP, I used the part I715039 and for Tet, I used the part J31007.

Once the digestion took place, Dr. Campbell notified me that heat inactivation was capable for Nco1 but not BamH1. There were 3 options for me to take in order to clean the Tet FML, the protocols for all of which can be found on the Davidson Lab Protocols link:

# [http://www.bio.davidson.edu/courses/Molbio/Protocols/Clean_Concentrate.html Clean and Concentrate DNA (after PCR, before digestion)]
# [http://www.bio.davidson.edu/courses/Molbio/Protocols/clean_short.html Ethanol Precipitate DNA (short protocol)]
# [http://www.bio.davidson.edu/courses/Molbio/Protocols/Qiagen_gelpure.html Qiagen QIAquick Gel Purification]

Although I could've chosen to do the heat inactivation for the RFP FML, I chose to keep the purifying technique in synch for consistency's sake. I chose to do the gel purification. While this process takes longer than the other two processes, I have found that gel purification generates the greatest yield and therefore chose it.

IGEM 2009 notebook

2009-06-18T20:43:37Z

ShPunjabi: /* Roclemente 14:00, 18 June 2009 (EDT) */

== [[User:Roclemente|Roclemente]] 16:54, 3 June 2009 (EDT) ==

Shamita and I are trying to find suitable reporter proteins to use. Yesterday, Leland and Alyndria were working on ways to insert the gene sequences into the plasmid. Upon seeing how they wanted to manipulated the reporter gene to include the logical clauses, we came up with a few criteria for the reporter genes we would use. The following criteria for genes are listed in order of the broadest aspect to look at to the narrowest aspect:

a) Doesn't contain restriction sites for the 4 restriction enzymes (EcoR1, Xbal, Spel, Pst1) used to cleave the Biobrick part out of the plasmid.

b) Contains 6 cutter restriction sites.

c) These restriction enzymes aren't blunt (cleave straight down at one spot).

d) These restriction sites are close to thge 5' (beginning) end of the sequence.

e) These enzymes are easiest to work with and cheapest.

We are finding the part numbers of the reporter genes we want to use (antibiotic resistance, fluorescence, ''LacZ'') through our own GCAT because we know these ones work. We are then locating these parts on the parts registry [http://www.partsregistry.org] website. We copied and pasted the gene sequences we obtained from the registry onto the ApE software [http://www.biology.utah.edu/jorgensen/wayned/ape/]. From here, we were able to generate a genetic map of each gene that outlined each restriction site that fit our criteria. We put each genetic map, alongside the part number used, into a Word document:

http://gcat.davidson.edu/GcatWiki/images/0/0e/Restriction_Site_Mapping_on_Reporter_Genes.doc

So it looks like we've changed directions. The team is looking towards the wet lab portion of our research more now. Instead of simply speculating about which reporter proteins we think will work best and which tRNA suppressors to use, we're going to physically test it out ourselves. A few tasks at hand in the second part of this afternoon:

1. What are the DNA gene sequences of the 12 different tRNA suppressors we want to use?

2. What is the sequence of the DNA that is cleaved off of both ends of the tRNA when it is transcribed?

We e-mailed Anderson to find out the exact tRNA gene sequences because we had a hard time finding this information everywhere else. Most papers we found describing the 5-base codon tRNA suppressors simply referred to Anderson's paper [http://gcat.davidson.edu/GcatWiki/images/6/6f/AndersonSchultz2002ChemBiol.pdf]. Anderson replied near the end of the day with the gene sequence. According to his email, the only difference between different tRNA genes is the anticodon loop. Therefore, if we just substitute all the anticodon loops in the invariable part of the sequence, we have our different tRNA sequences. We plan on using the Lancilator to help us break these tRNA sequences into smaller oligonucleotides that can anneal at around the same temperature because it is not possible for us to order oligos that are greater than 70 nucleotides long.

== [[User:Roclemente|Roclemente]] 17:21, 4 June 2009 (EDT) ==

I started out the day looking at my time-sensitive group research qustion about reporter proteins. I started making a table listing the advantages and disadvantages of different reporter proteins. Then the group found out that there was a major roadblock to our project: a stop codon was located in our ATG-5mer oligo. The stop codon would have been part of the BioBrick scar. The group looked at several ways to work around this problem. The idea that I looked most into was replacing the restriction sites that were used in the scar through standard assembly. According to the judging criteria for iGEM, I found that a team could possibly alter the standard assembly method as long as they wrote a letter to the iGEM judges explaining our plan in a detailed manner. From there, I set off to understand the BioBrick scar more by making a document highlighting the exact positions of the rsetriction sites on the BioBrick parts and how they were used to put more than one part together. I then found several other pairs of restriction sites that complemented one another and could be used in place of the standard scar between Xba1 and Spe1. At the end of the day, the group got the chance to speak with Dr. Campbell and he suggested a hybrid idea which would combine Davidson's restriction site idea with Missouri's PCR idea. I'm not quite sure of what this idea is exactly yet, but we'll all clarify our vague idea of it tomorrow morning after a talk with Dr. Eckdahl.

== [[User:Roclemente|Roclemente]] 17:13, 5 June 2009 (EDT) ==

This morning we began exploring different options for bypassing the stop codon problem from yesterday. If we wanted to make sure we were using the BioBrick parts in our GCatalog, there was no way we were going to be getting rid of the Xba1 restriction site that contained the "TAG" which codes for a stop codon. Therefore, we proposed looking at restriction sites within the reporter genes instead, therefore addressing the already-placed BioBrick ends. Using the document me and Shamita created on "Restriction Site Mapping on Reporter Genes" (see first journal entry), we determined that the reporter gene with the restriction site closest to the beginning of the sequence was YFP. We proposed cutting the YFP gene that we already had in stock at this restriction site and inserting an oligo (which we would have ordered) with a sequence in the following order: "BioBrick prefix - ATG - 5mer - part of YFP gene to the left of the cleaved restriction site."

After clarifying our own idea with one another, we spoke with some Missouri kids to clarify the said "hybrid" idea which, according to Dr. Campbell, combined the ideas of both campuses. The hybrid idea was based upon our idea of cutting the reporter gene at a restriction site as well, except that our primer would start out with a primer for our reporter gene, and to the left of the primer would be a "tail" consisting of the ATG and 5mer and either an Xba1 or EcoR1 restriction site (We still have to decide this. Our decision will probably be based on the restriction site we end up using in our reporter gene and whether or not that is complementary to either the Xba1 or EcoR1 sites.). We would use the PCR technique and use our in-stock reporter gene strand as our template strand (it will be denatured into single strands) and we will add our oligo. We propose that the primer will synthesize the rest of the YFP strand as well as add on nucleotides to complete the oligo tail. In the end, we will have a double stranded DNA sequence consisting of either the Xba1 or EcoR1 site, the ATG, the 5mer, and the reporter gene. The beauty of this hybrid idea is that we have flexibility as far as the reporter gene we choose to use since all we really need for it is the beginning sequence (for the primer) and a known, "hearty" restriction site. Once we cut this double strand at this restriction site on the reporter gene as well as at the Xba1 or EcoR1 sites, we can insert this into a plasmid that contains the rest of that reporter gene after the restriction site we choose to use and the complementary Xba1 or EcoR1 site.

I made a Word document outlining the promoter and RBS parts we have in our registry on the Davidson campus. Leland and I went through the registry parts on the GCATalog to look for potential promoters and RBS parts. We went through the document as a group and analyzed each of these potential parts on partsregistry.org to see which ones would most likely work the best in the miniprep we'll start preparing for on Sunday evening. Here is the document we wound up with:

http://gcat.davidson.edu/GcatWiki/images/d/d2/Promoters_and_RBS.doc

== [[User:Roclemente|Roclemente]] 10:40, 8 June 2009 (EDT) ==

We're starting the day by finalizing the oligos we will be ordering to have synthesized. Two groups of oligos must be sequenced: primers and tRNA gene sequence. The primers will consist of a forward and reverse primer. The forward primer will consist of the following in this 5' - 3' order: GCAT - BioBrick Prefix - ATF - 5mer - first 20 nucleotides in the reporter gene sequence. These 20 nucleotides won't include the ATG that naturally comes at the beginning of the reporter gene because we want to make sure the ribosome attaches to the first ATG and reads the logical clause instead of going straight to the ATG at the start of the reporter gene. The reverse primer will consist of the first 20 nucleotides downstream of the restriction site we plan to use in the reporter gene. I worked on finding the primers for the Tet resistance gene (part J31007) while Lyn worked on finding the primers for the RGP gene. Dr. Campbell decided we should use the BamH1 restriction enzyme because it is very "hardy." The following document outlines the 5 forward primers (each with one of the five variable 5mers) we will order for amplification of the Tet resistance FML:

[[Image:Tet resistance forward primers.png]]

This is the reverse primer we will order for the Tet resistance FML:

[[Image:Tet resistsance reverse primer.png]]

== [[User:Roclemente|Roclemente]] 11:54, 9 June 2009 (EDT) ==

We need to digest the plasmids at the EcoR1 and Pst siets. Everytime we're going to set up a digestion, we'll want the following in these volumes: 10x Buffer (2 uL), DNA (~ 3 uL), enzymes 1 and 2 (1 uL each), water (13 uL), and final volume of all of these put together should be 20 uL.

Some notes about digestion: Enzymes should make up 10% of the final volume. As a general rule of thumb, make sure the tip of your pipet is close to the surface of the solution. This is especially pertinent when gathering 1 microL of each of the enzymes. The most sensitive components are the enzymes, so they should go last.

We'll have a bunch of tubes with each 3 microL of the specific DNA. We'll make a cocktail in a bigger tube consisting of the buffer, 2 enzymes, and water. The rationale behind thissis that you'll be less likely to make a bigger mistake with a bigger volume (versus getting 1 microL of enzyme per specific DNA). You'll also know that there is no problem with the enzyme if all the DNA don't cut. If only one specific DNA doesn't work, then we'll know that it's because there's something wrong with the specific DNA rather than anything in the "Master Mix." Since we have to do 11 digestions, we'll make enough of the master mix for 12 tubes to make sure we have enough. We'll take 17 microL of the Master Mix to each tube of DNA.

The optimal temperature for most enzymes is 37 degreees celsius. You'd like to have enzymes with the same optimal temperatures (for ex. restriction enzymes that start with B usually have an optimal temperature near 50 degrees. You wouldn't want to use both B-enzyme and EcoR1 because EcoR1 would die).

'''What percent gel should we use?'''

Look at the Davidson lab protocol on the Davidson-Missouri Wiki. Generally, bigger molecules require an agarose gel with a lower concentration.

'''What's next in the future?'''

5' end additions are done by PCR. We'll ampliffy this double-stranded DNA. We'll cut with both EcoR1 and BamH1 or Nco1 (depending on which reporter gene you're using). Then we'll cut the plasmid with the reporter with the same restriction enzymes used.Then, to throw away the remaining DNA that we don't want, we'll run a gel and throw away the smaller band (because the larger band is probably made of the plasmid we want to keep).Then we'll take the double stranded DNA we amplified and the purified plasmid and ligate, transform, and screen them and verify the sequence.

While we wait for the oligos, we can cut the plasmid with reporter on the same restriction enzyme already.

'''How are we building the tRNAs?'''

We'll assemble the oligos into genes by mixing our oligos together, boiling them, and very slowly allowing them to cool. This will require many calculations. We'll cut the plasmids with EcoR1 and Pst1. to be ready to receive the enes we're assembling.We'll have to gel purify the plasmids to get them away from their inserts. We'll ligate, transform, and screen the genes and the plasmids and verify the sequence. On paper you shouldn't have two sites cut with different restriction enzymes stick together, but if there's selection pressure, these sites may cut some ends to fit together. This is why we must screen them.

After Dr. Campbell taught us all of the above, the 5 biologists put together a document outlining exactly how we would go about cutting the two sets of plasmids (one set will be cut with Eco R1 and Pst1 and othe other set will be cut with Eco R1 and BamH1 or Nco1). The following table outlines which biologist was going to work with which sample of DNA:

[[Image:Names and DNA.jpg]]

The following summarize the diferent materials and volumes of these materials that we'll need for our digestion of the plasmids:

[[Image:Digestion materials.png]]

This last table highlights the agarose percent concentration we should use for each part according to how many base pairs it contains:

[[Image:Screen-capture-3.png]]

The biologists digested the plasmids that we plan on using once our oligos arrive in a day or two. Afterwards, we made the two agarose gels that were most compatible with the parts we were going to run. This gel is going to purify the plasmids so that we have the parts that we're going to use later on, minus the rest of the DNA in the plasmid that we don't need. Before physically creating the gel, we figured out how much agarose we would need to make our two different agarose gel concentrations, 0.8% and 2.5%. Next, we made sure the mold and the big teeth were in place. We used the big teeth at the end of the mold because we plan on running 20 uL of DNA versus a running a smaller portion which would require us to use small teeth. We then measured the amount of agarose that we calculated we would need, placed this in a volumetric flask greater than 60 uL to allow for fizz to rise up, then measured out 60 uL of buffer and mixed this in the same flask with the agarose. We placed clear plastic wrap on top of the flask and heated it up for 1 minute and 20 seconds in the microwave. After we heated it, we took it out and checked to see if there was still agarose present that hadn't yet melted. There was, so we placed it back in the microwave and heated it for another 20 seconds. All the agarose had melted. We added 1 uL of EtBr (ethidium bromide) to the solution and quickly poured this into the mold. It needs about 15 to 20 minutes to cool. Dr. Campbell made the agarose gel that was 2.5% concentrated gel while we watched. We then made the 0.8% concentrated gel while he watched.

The following table lists the order in which we loaded our DNA into the gel. The first column all the way to the left is represented by number 1:

[[Image:Screen-capture-2.png]]

== [[User:Roclemente|Roclemente]] 08:46, 10 June 2009 (EDT) ==

After about 30 minutes after we began the gel electrophoresis, we looked at our gels with UV light to see how far the inserts had travelled and where the plasmids were. Then we took each gel to be photographed in the freezer room (231). We used the following DNA length marker to measure how long each sequence represented by each band was:

[[Image:Screen-capture-1.png]]

We compared our data to the lengths we expected to get according to the registry online. The first gel we photographed was the one that was 2.5% concentrated:

[[Image:2009-06-09 14hr 44min.jpg]]

The first two columns of the DNA we ran, both consisting of B0300, were fairly the same as the length we expected. However, the last two columns, consisting of K091111 and K091112 respectively were supposed to be 86 bp long, however, according to our data, K091112 was longer. Leland and I looked at Pallavi Penumetcha's paper which delved more closely into these two parts and what she wrote in her paper and what she entered into the registry conflict. We are currently corresponding with her to figure out the exact discrepancy.

We then photographed the 0.8% gel:

[[Image:2009-06-09 15hr 21min.jpg]]

The length of the sequences in almost every column matched up with the lengths we expected. Only the 4th and 5th columns didn't come out clearly. We think that the DNA inserted in column 5 weren't inserted properly and diffused on its own. We couldn't see bands in the 4th column at all until we adjusted the color a bit on the photographer and saw that column 4 was pretty much identical to column 5. We assumed that column 5 bled into column 4.

== [[User:Roclemente|Roclemente]] 15:14, 11 June 2009 (EDT) ==

We are digesting two parts today: I715039 (pLac+RBS+RFP) and S03710 (Tet Resistance). We want both the plasmids and the inserts in each case. We are aiming for a final volume of 40 uL for each part. This means that each digestion will consist of 4 uL buffer, 2 uL EcoR1, and 2 uL of their other respective restriction site (BamH1 for S03710 and Nco1 for I715039). The only parts that will vary are the amount of DNA and water we use. The amount of DNA we use depends on the concentration of the DNA we have (found through use of the NanoDrop). Since we want 2500 ng of DNA, we can use the concentration of DNA to figure out how much volume of this concentrated DNA we'll want to use in your digestion. If we subtract the volumes of buffer, two restriction enzymes, and DNA we plan on using from 40 uL, we'll have the amount of water we need to add. Here are the different volumes of materials we plan on using in our double digestion:

[[Image:DoubledigestRFPTetR.png]]

Now that we know how much of each material we were going to use, we needed to find out which buffer would be compatible with each sample according to the restriction enzymes we were using and how effective they'd be in different salt solutions. Online on the Promega "Compatible Buffers" website, we were able to find which Promega buffers would be most compatible when doing a double digest with EcoR1 and BamH1:

[[Image:BamEcoR1.png]]

Here is a chart noting buffers and their compatiblity with EcoR1 and Nco1:

[[Image:NcoEcoR1.png]]

We still have to wait for the restriction enzymes to arrive, so afterwards, we transformed three different inserts containing needed promoters and RBSs into vectors. We used the Zippy Transformation method. These promoters and RBSs will later be added to the FMLs once they are already inserted into the reporter cells. The following table outlines these inserts and part numbers:

[[Image:Promoterinsertspartnumbers.png]]

The restriction enzymes have arrived. We will now be able to do PCR to amplify the RFP gene (I715039) and the Tet resistance gene (S03710) as templates. We will be using the primers which have also arrived. We ordered forward and reverse primers so that our amplified sequence will consist of the following in this order:

GCAT-ATG-5mer-Reporter Gene

The following table lists the materials will be needed for this PCR technique which uses the "Green Promega master (Monster) Mix:"

[[Image:Greenpromegamastermix.png]]

This is the PCR procedure:

[[Image:Temperaturecycle.png]]

These are the variables that we will be adding to each separate sequence we want to be amplified:

[[Image:Tobeamplified.png]]

This table shows how we labeled the tubes based on how we divided the PCR work for the 10 tubes:

[[Image:Pcrtubes.png]]

Now we are giong to assemble the tRNA oligos together to form the tRNA genes. We could only order a certain length of sequence and the tRNA genes outdid these specified lengths. We used the Lancilator to find various oligonucleotides that overlapped and would be able to anneal together to form the tRNA genes. We used the Davidson lab protocol entitled "Buiding dsDNA with Oligos" to assemble the oligos:

[[Image:Oligoassembly.png]]

== [[User:Roclemente|Roclemente]] 22:46, 12 June 2009 (EDT) ==

We had two main goals for today:
1. perform a tRNA ligation and transformation
2. perform two electrophoreses: one to verify the PCR sequences we obtained and another to purify the digested reporter plasmids we would need

We began the day by pouring the agarose gels according to which concentration of buffer would be most compatible with the molecular weight of the substance we wanted to isolate. For both the digestion of E01010 and J31007, according to the "Optimize your Gel" website found on the Davidson Lab Protocols site for iGEM 2009, we wanted a 0.4% concentrated gel. For the PCRs, we wanted a 1.6% concentrated gel to optimize our retrieval of the Tet gene and a 1.4% concentrated gel to optimize our retrieval of the RFP gene.

While we waited for the gels to harden for about 30 minutes, we began calculating how much of each material we would need to ligate our tRNAs. We used the following equation to find out how much insert we should use:

[[Image:Ligationequation.png]]

Using the lengths of sequences we would be ligating, this is the mass that we needed:

X nanograms of insert = [(2)(144bp)(50 ng linearalized plasmid)]/2038bp
X = 7.0657 ng of insert

The following table outlines our calculations for finding the mass of tRNAs we had:

[[Image:Ligationcalculations.png]]

We would attain this mass of tRNA by pipetting an unknown amount of it. In order to attain a more "pipetable" amount of tRNA, we diluted our initial concentration 50X. Since we knew we wanted our final mass to be 7.065 ng, we calculated the volume we would need to take from this new, diluted sample:

DILUTION: 555.48/50 = 11.012
CALCULATION: 11.012 ng/ 1uL = 7.065 ng/ Y uL
Y = .64 uL of insert

Afterwards, Olivia and Alyndria began the actual ligation process while Shamita, Leland, and I filled the wells of the two gels. The first included the two digested reporter parts. We also included a molecular weight marker. Here is a photograph of this gel:

[[Image:2009-06-12 15hr 54min.jpg]]

According to our prior calculations, the Tet plasmid should have been 2958 base pairs long and the RFP plasmid should have been 2380 base pairs long. Our expectations were verified by the data. Shamita and I then sliced the part of thsi gel containing both plasmids and put these slices away to purfiy on Monday.

The second gel contained aliquots of the PCR we had done to attain 10 FML sequences: 5 containing the Tet resistant reporter gene and each of the 5 frameshifts that Davidson was assigned and the other 5 containing the RFP reporter gene and each of the 5 frameshifts as well:

[[Image:2009-06-12 16hr 07min.jpg]]

According to our data, each sequence containing the Tet resistant gene should have been 317 base pairs long and each sequence containing the RFP gene should have been 446 base pairs long. Our data supports our hypothesis, however, the 3rd well was not successful. This well contained the FML consisting of the Tet resistant gene and the frameshift mutant CUAGU. Before leaving work for the day, Shamita and I prepared and performed PCR using two parts containing the tet resistant gene, J31007 and S03710. We wanted to use both to make sure at least one of them works since we could technically use either of them, even though when we first did PCR we only used the S03710 part. Using the known concentrations of each of these parts, and knowing that we needed 1 ng of each part to include in our PCR samples, we calculated the volume of each 100X-diluted solution we would need:

S03710: 1.636 ng/uL = 1 ng/ X uL
X = 0.611 uL

J30017: 0.776 ng/uL = 1 ng/ Y uL
Y = 1.289 uL

We will run another gel with both of these parts on Monday and hopefully at least one of them works.

== [[User:Roclemente|Roclemente]] 17:24, 15 June 2009 (EDT) ==

Each biology member of the Davidson iGEM team has been assigned a 5mer to carry on throughout the rest of their respective processes. I've been assigned the 5mer CUAGU. We verified the PCR of the CUAGU FML containing the RFP gene on Friday. However, we weren't able to verify the CUAGU FML containing the tet resistance gene when we ran it on a gel. Shamita and I redid PCR for two parts containing the tet resistance gene: J31007 and S03710, labeled 2* and 2! respectively. I'm running another gel to verify either or both of them. Both sequences should be 317 bp long (this includes the ATF, logical clause, and the tet resistance gene to the left of the BamH1 restriction site). Here is the gel run we collected. The first column represents the 1 kb DNA marker, and the next two are 2* then 2!:

[[Image:2009-06-15 10hr 34min.jpg]]

The data shows that the PCRs of both parts wound up at the length we expected. The first time we did PCR on J31007, we must have left a solution out of our procedure on accident. I combined both PCRs together since they are the same length and match the length we expected to get.

Next, we cleaned and concentrated our amplified DNA sequence (#D4014 from Zymo Research). I cleaned the two pieces of DNA that contained the 5mer CUAGU. I labeled the tube with DNA also containing the RFP gene as "1" and the tube with DNA also containing the tet resistance gene as "2!". We needed 2 volumes of buffer for every 1 volume of DNA we added. Therefore, for tube "1," I added 95 uL DNA and 190 uL buffer. I added more DNA to tube "2!" since I combined the DNA from two PCR events to one (2* and 2! from earlier).

After cleaning the DNA, we found its concentration using the Nanodrop. In a solution made up of 0.5 uL of the DNA and 2.5 uL of TE, we found the following data from the DNA in this solution:

[[Image:Cleanconcentrate.png]]

The concentration of the DNA we cleaned was the concentration given by the Nanodrop multiplied by 6 (the DNA was dilute 6-fold for the Nanodrop). We used this concentration and the volume of clean DNA we knew we had (9.5 uL) to find the mass of clean DNA we had:

[[Image:Concentrateddna.png]]

Before doing digestion, we used the ligation equation (see "Ligation Protocol" in "Davidson Lab Protocols") to figure out how much mass of the DNA insert we would need later on when we did ligation. For the RFP FML, we found we would want the following mass of insert:

ng of insert = (2)(443)(50)/(2320) = 19.0948 ng

We multiplied this mass by 50 because we're counting on losing DNA between the digestion and cleaning we would need to do leading up to the ligation protocol.

19.0948 ng x 50 = 954.741 ng RFP FML

We found the mass of Tet FML insert we would need for ligation as well:

ng of insert = (2)(314)(50)/(2958) = 10.6153 ng

We multiplied this mass by 50 too:

10.6153 x 50 = 530.764 ng Tet FML

Unfortunately, nobody had enough of their clean DNA to satisfy our careful criteria. However, we would need to do this calculation later on so it was helpful we did it anyway. Instead, we just used all the DNA we had, or 9.5 uL for each reporter gene. Since there were 4 people who needed the same master mix, we made 5 parts of the master mix using the following volumes:

[[Image:Rfptetdigestion.png]]

After realizing that we would have to wait a day for the digestion to take place, we decided to culture every tRNA cell that colonized. The tRNA that we transformed on Friday did not have a good yield, so last night a few people came in and transformed more tRNA cells. We got a few more colonies. To be safe and make sure we cultured each tRNA, we picked every colony visible to us in both the old and new plates. Using our own given tRNAs, I counted the amount of colonies that grew in the plate we transformed on 6/12 and 6/14. There were 4 colonies that grew on both plates so I labeled a tube for each of these. I awas also in charge of the negative control. There were 2 colonies that grew on the negative control plate so I labeled 2 tubes for that. We will be placing them in an incubator overnight.

== [[User:Roclemente|Roclemente]] 12:30, 16 June 2009 (EDT) ==

We checked on the cells we cultured overnight. Of the cells that I cultured (all containing the CUAGU 5mer and the two negative controls), both negative control tubes worked as well as the tubes I labeled 1 (6/14), 5 (6/12), and 7 (6/12). The rest of mine were either red, meaning the plasmids hadn't ligated properly) or the colonies we used were no good. I took these 5 tubes, along with 3 of Olivia's tubes filled with cells that were cultured (tubes 11, 12, and 13) and miniprepped them to isolate the plasmids from the cells. There was no need to colony PCR to screen for successful ligations because this is only used to know which ones to let grow overnight and we had already done this.

As soon as I finished miniprepping them, I had to leave the lab for a little bit, so Olivia took each my miniprep tubes besides the negative controls and Leland took my negative controls. I completed the protocol for digestion of the tRNAs with EcoR1 and Pst1 for Alyndria's tubes when I got back because she had left a little earlier for lunch.

Afterwards, Shamita and I poured a 1.9% concentrated gel to load the digested tRNAs on. We want to verify that these tRNAs are the appropriate size for if they were cut properly. Since we will have to load 37 wells, and the maximum number we can load on one gel is 18 since 2 wells will have to be used by the molecular weight marker, we needed to pour 2 gels and we will also be using a half of a 2.0% agarose gel that was left over from another experiment.

Shamita and I also made new 0.5X running buffer gel. We did this by adding 450 uL buffer and 9 uL ethidium bromide (1 uL EtBr : 50 uL buffer). Alyndria, Leland, and Shamita loaded these gels.

Olivia and I cleaned our PCR DNA using the "Ethanol precipitate DNA (short protocol)" found in the Davidson Lab Protocols link. To begin with, I added 180 uL dH2O to my two samples, CUAGU with RFP and CUAGU with Tet, to bring their volumes up to 200 uL. During one of the last steps of this protocol, when Olivia and I dried out our DNA using the SpeedVac for a period of 10 minutes, the top of my tube containing the CUAGU 5mer and RFP gene broke. I wasn't sure if a pellet was in there, but I still poured out the ethanol and went on with the protocol. At the end, I resuspended what may or may not have been my DNA in 10 uL distilled water. I NanoDropped it afterward and attained the following data concerning both tubes:

[[Image:Failedpcrmeh.png]]

According to this data, any absorption and concentration we may have attained is insignificant because the A-260 must be between 0.05 and 1.50 to be significant and neither of the DNA is. Tomorrow I will do PCR for both FMLs over again.

== [[User:Roclemente|Roclemente]] 14:51, 17 June 2009 (EDT) ==

I found out that my tRNA DNA that I labeled "1" and which contains CUAGU was verified from the gel run Olivia did yesterday (the gel run also included her tRNA DNA samples). At the start of the day I plated this tube of cultured cells again to grow overnight.

Since PCR will take about 3 1/2 hours, I attempted this procedure at the beginning of the day. I plan on making 5 PCR products for each reporter gene since I now know the environmental conditions are correct. I labeled the RFP tubes 1-5 and the Tet tubes 6-10. I first needed to figure out how much of the template DNA I would need in order to have 1 ng template DNA. I made the following calculations to find this out:

[[Image:1ngtemplatedna.png]]

I wanted to add the Monster Mix last, so I made the following table, adding everything else besides the Monster Mix for 5 of the same reporter gene to one tube:

[[Image:Pcrmix.png]]

Unfortunately, when I actually mixed the solution together, I forgot to dilute the template DNA. Therefore, we have 100X more template DNA than we would have had we diluted the template DNA. The PCR can still work, but we will have to constantly remind ourselves that there will be extra template DNA in our solution and that this may skew, however slightly, our results later on.

While I waited for the PCR product, I refocused on getting my tRNA DNA ready for sequencing. Using the NanoDrop, I found the concentration of my miniprepped CUAGU DNA that was verified to be 61 ng/uL. The pre-sequencing protocol calls for 60 ng DNA, so I extracted 1 uL from this tube (the extra DNA will be insignificant in the sequencing procedure). I pipetted this into another tube and used the SpeedVac to dry it out. This is how we will send our DNA to be sequenced.

The following is a summary of steps I put together to help me plan out what I will do after the PCR procedure is over (it will be done at 5 pm today):

1. Combine the five matching tubes into one tube.
2. Run each PCR product on a gel to verify that our expected sequence has been properly amplified.
3. Clean and concentrate plasmids so salt solution is optimal for digestion.
4. Double digest PCR products with EcoR1 and BamH1 or Nco1.
5. Clean and concentrate again to get rid of restriction enzymes.
6. Nanodrop inserts in order to do ligation calculations and verify that sequences are present.
7. Ligate FML inserts with purified plasmids.
8. Transform plasmid into cells.

Since I won't have time to run a gel and take a picture once the PCR procedure is complete (we get out at 5:30 and it will be finished at 5:00), I have begun to prepare the agarose gel for use tomorrow. The size of the band I expect to find for the Tet FML is 349 bp (including the GCAT, BioBrick prefix, ATG, 5mer, and reporter gene until about 20 nucleotides or so past the restriction site we will cut at) and 474 bp for the RFP FML. According to the "Optimize Your Gel" link on the Davidson Lab Protocols page, a 1.6% gel will optimize how well I see my Tet bands and a 1.3% gel will optimize how well I see my RFP bands. I can use a gel with a concentration in between both of the optimal concentrations for Tet and RFP so that I can use one gel for both. Therefore, I will use a 1.45% gel. 1.45% of 60 uL buffer solution tells me that I should measure out 0.87 g agar for this gel.

== [[User:Roclemente|Roclemente]] 14:00, 18 June 2009 (EDT) ==

I checked on my PCR products this morning. Tubes 1 and 2 (both containing the RFP FML) had solution scattered on the tube's cover and walls so I placed them in the centrifuge for a bit. Unfortunately, I forgot to put these 500 uL tubes in holders when I centrifuged them so these tubes were destroyed. This was okay though because I was just going to combine each respective FML into one tube. I would have 300 uL of RFP FML PCR product instead of 500 uL and I put all of this in tube 5. I still had 500 uL of Tet FML PCR product left and put all of this in tube 10.

I ran 5 uL of both RFP and Tet FMLs on a gel, as well as a 1 kb molecular weight marker. The MW marker is in lane 1, the RFP is in lane 3, and the Tet is in lane 4 (Leland ran his PCR product in lane 2):

[[Image:2009-06-18 09hr 56min.jpg|thumb|none|500px]]

I was expecting the RFP PCR product to be 474 bp long and the Tet PCR product to be 349 bp long. Both appear to come out a bit shorter, but Dr. Campbell and I agreed that we wouldn't count these sequences out because the bands were quite close to what we expected.

I proceeded to clean and concentrate these two sequences using the procedure outlined in the "Clean and Concentrate DNA (after PCR, before digestion)." Now I had 295 uL of the Tet sequence and 495 uL of the RFP sequence. Since the procedure says I can only have 800 uL in the column at a time, with DNA:Buffer at a ratio of 1:2, I mixed the two in a column in a series of two rounds using the following volumes:

[[Image:Clean.png]]

I Nanodropped my DNA after cleaning it and attained the following results:

[[Image:Nanome.png]]

I wanted the OD260 (Optical Density at 260) to be under 1.5, which it is for both. I lost quite a bit of Tet DNA in the process, and will only continue to lose DNA. To account for this loss of DNA, Dr. Campbell suggested it would be a good idea to heat inactivate the restriction enzymes to get rid of them after digestion instead of cleaning and concentrating the DNA over again.

I double digested both RFP and Tet FML sequences. I used all 9.5 uL of my DNA. The following table outlines how much volume of each material I mixed together:

[[Image:2nddoubledigest.png]]

While I waited 3 hours for my digestion to take place, I prepared 6 tubes to make more liquid cultures of the RFP and Tet vectors to grow more receiving vectors. 3 tubes would be to make RFP cultures and the other 3 would be for the Tet cultures. For RFP, I used the part I715039 and for Tet, I used the part J31007.

Once the digestion took place, Dr. Campbell notified me that heat inactivation was capable for Nco1 but not BamH1. There were 3 options for me to take in order to clean the Tet FML, the protocols for all of which can be found on the Davidson Lab Protocols link:

# [http://www.bio.davidson.edu/courses/Molbio/Protocols/Clean_Concentrate.html Clean and Concentrate DNA (after PCR, before digestion)]
# [http://www.bio.davidson.edu/courses/Molbio/Protocols/clean_short.html Ethanol Precipitate DNA (short protocol)]
# [http://www.bio.davidson.edu/courses/Molbio/Protocols/Qiagen_gelpure.html Qiagen QIAquick Gel Purification]

Although I could've chosen to do the heat inactivation for the RFP FML, I chose to keep the purifying technique in synch for consistency's sake. I chose to do the gel purification. While this process takes longer than the other two processes, I have found that gel purification generates the greatest yield and therefore chose it.

IGEM 2009 notebook

2009-06-18T20:43:19Z

ShPunjabi: /* Roclemente 14:00, 18 June 2009 (EDT) */

== [[User:Roclemente|Roclemente]] 16:54, 3 June 2009 (EDT) ==

Shamita and I are trying to find suitable reporter proteins to use. Yesterday, Leland and Alyndria were working on ways to insert the gene sequences into the plasmid. Upon seeing how they wanted to manipulated the reporter gene to include the logical clauses, we came up with a few criteria for the reporter genes we would use. The following criteria for genes are listed in order of the broadest aspect to look at to the narrowest aspect:

a) Doesn't contain restriction sites for the 4 restriction enzymes (EcoR1, Xbal, Spel, Pst1) used to cleave the Biobrick part out of the plasmid.

b) Contains 6 cutter restriction sites.

c) These restriction enzymes aren't blunt (cleave straight down at one spot).

d) These restriction sites are close to thge 5' (beginning) end of the sequence.

e) These enzymes are easiest to work with and cheapest.

We are finding the part numbers of the reporter genes we want to use (antibiotic resistance, fluorescence, ''LacZ'') through our own GCAT because we know these ones work. We are then locating these parts on the parts registry [http://www.partsregistry.org] website. We copied and pasted the gene sequences we obtained from the registry onto the ApE software [http://www.biology.utah.edu/jorgensen/wayned/ape/]. From here, we were able to generate a genetic map of each gene that outlined each restriction site that fit our criteria. We put each genetic map, alongside the part number used, into a Word document:

http://gcat.davidson.edu/GcatWiki/images/0/0e/Restriction_Site_Mapping_on_Reporter_Genes.doc

So it looks like we've changed directions. The team is looking towards the wet lab portion of our research more now. Instead of simply speculating about which reporter proteins we think will work best and which tRNA suppressors to use, we're going to physically test it out ourselves. A few tasks at hand in the second part of this afternoon:

1. What are the DNA gene sequences of the 12 different tRNA suppressors we want to use?

2. What is the sequence of the DNA that is cleaved off of both ends of the tRNA when it is transcribed?

We e-mailed Anderson to find out the exact tRNA gene sequences because we had a hard time finding this information everywhere else. Most papers we found describing the 5-base codon tRNA suppressors simply referred to Anderson's paper [http://gcat.davidson.edu/GcatWiki/images/6/6f/AndersonSchultz2002ChemBiol.pdf]. Anderson replied near the end of the day with the gene sequence. According to his email, the only difference between different tRNA genes is the anticodon loop. Therefore, if we just substitute all the anticodon loops in the invariable part of the sequence, we have our different tRNA sequences. We plan on using the Lancilator to help us break these tRNA sequences into smaller oligonucleotides that can anneal at around the same temperature because it is not possible for us to order oligos that are greater than 70 nucleotides long.

== [[User:Roclemente|Roclemente]] 17:21, 4 June 2009 (EDT) ==

I started out the day looking at my time-sensitive group research qustion about reporter proteins. I started making a table listing the advantages and disadvantages of different reporter proteins. Then the group found out that there was a major roadblock to our project: a stop codon was located in our ATG-5mer oligo. The stop codon would have been part of the BioBrick scar. The group looked at several ways to work around this problem. The idea that I looked most into was replacing the restriction sites that were used in the scar through standard assembly. According to the judging criteria for iGEM, I found that a team could possibly alter the standard assembly method as long as they wrote a letter to the iGEM judges explaining our plan in a detailed manner. From there, I set off to understand the BioBrick scar more by making a document highlighting the exact positions of the rsetriction sites on the BioBrick parts and how they were used to put more than one part together. I then found several other pairs of restriction sites that complemented one another and could be used in place of the standard scar between Xba1 and Spe1. At the end of the day, the group got the chance to speak with Dr. Campbell and he suggested a hybrid idea which would combine Davidson's restriction site idea with Missouri's PCR idea. I'm not quite sure of what this idea is exactly yet, but we'll all clarify our vague idea of it tomorrow morning after a talk with Dr. Eckdahl.

== [[User:Roclemente|Roclemente]] 17:13, 5 June 2009 (EDT) ==

This morning we began exploring different options for bypassing the stop codon problem from yesterday. If we wanted to make sure we were using the BioBrick parts in our GCatalog, there was no way we were going to be getting rid of the Xba1 restriction site that contained the "TAG" which codes for a stop codon. Therefore, we proposed looking at restriction sites within the reporter genes instead, therefore addressing the already-placed BioBrick ends. Using the document me and Shamita created on "Restriction Site Mapping on Reporter Genes" (see first journal entry), we determined that the reporter gene with the restriction site closest to the beginning of the sequence was YFP. We proposed cutting the YFP gene that we already had in stock at this restriction site and inserting an oligo (which we would have ordered) with a sequence in the following order: "BioBrick prefix - ATG - 5mer - part of YFP gene to the left of the cleaved restriction site."

After clarifying our own idea with one another, we spoke with some Missouri kids to clarify the said "hybrid" idea which, according to Dr. Campbell, combined the ideas of both campuses. The hybrid idea was based upon our idea of cutting the reporter gene at a restriction site as well, except that our primer would start out with a primer for our reporter gene, and to the left of the primer would be a "tail" consisting of the ATG and 5mer and either an Xba1 or EcoR1 restriction site (We still have to decide this. Our decision will probably be based on the restriction site we end up using in our reporter gene and whether or not that is complementary to either the Xba1 or EcoR1 sites.). We would use the PCR technique and use our in-stock reporter gene strand as our template strand (it will be denatured into single strands) and we will add our oligo. We propose that the primer will synthesize the rest of the YFP strand as well as add on nucleotides to complete the oligo tail. In the end, we will have a double stranded DNA sequence consisting of either the Xba1 or EcoR1 site, the ATG, the 5mer, and the reporter gene. The beauty of this hybrid idea is that we have flexibility as far as the reporter gene we choose to use since all we really need for it is the beginning sequence (for the primer) and a known, "hearty" restriction site. Once we cut this double strand at this restriction site on the reporter gene as well as at the Xba1 or EcoR1 sites, we can insert this into a plasmid that contains the rest of that reporter gene after the restriction site we choose to use and the complementary Xba1 or EcoR1 site.

I made a Word document outlining the promoter and RBS parts we have in our registry on the Davidson campus. Leland and I went through the registry parts on the GCATalog to look for potential promoters and RBS parts. We went through the document as a group and analyzed each of these potential parts on partsregistry.org to see which ones would most likely work the best in the miniprep we'll start preparing for on Sunday evening. Here is the document we wound up with:

http://gcat.davidson.edu/GcatWiki/images/d/d2/Promoters_and_RBS.doc

== [[User:Roclemente|Roclemente]] 10:40, 8 June 2009 (EDT) ==

We're starting the day by finalizing the oligos we will be ordering to have synthesized. Two groups of oligos must be sequenced: primers and tRNA gene sequence. The primers will consist of a forward and reverse primer. The forward primer will consist of the following in this 5' - 3' order: GCAT - BioBrick Prefix - ATF - 5mer - first 20 nucleotides in the reporter gene sequence. These 20 nucleotides won't include the ATG that naturally comes at the beginning of the reporter gene because we want to make sure the ribosome attaches to the first ATG and reads the logical clause instead of going straight to the ATG at the start of the reporter gene. The reverse primer will consist of the first 20 nucleotides downstream of the restriction site we plan to use in the reporter gene. I worked on finding the primers for the Tet resistance gene (part J31007) while Lyn worked on finding the primers for the RGP gene. Dr. Campbell decided we should use the BamH1 restriction enzyme because it is very "hardy." The following document outlines the 5 forward primers (each with one of the five variable 5mers) we will order for amplification of the Tet resistance FML:

[[Image:Tet resistance forward primers.png]]

This is the reverse primer we will order for the Tet resistance FML:

[[Image:Tet resistsance reverse primer.png]]

== [[User:Roclemente|Roclemente]] 11:54, 9 June 2009 (EDT) ==

We need to digest the plasmids at the EcoR1 and Pst siets. Everytime we're going to set up a digestion, we'll want the following in these volumes: 10x Buffer (2 uL), DNA (~ 3 uL), enzymes 1 and 2 (1 uL each), water (13 uL), and final volume of all of these put together should be 20 uL.

Some notes about digestion: Enzymes should make up 10% of the final volume. As a general rule of thumb, make sure the tip of your pipet is close to the surface of the solution. This is especially pertinent when gathering 1 microL of each of the enzymes. The most sensitive components are the enzymes, so they should go last.

We'll have a bunch of tubes with each 3 microL of the specific DNA. We'll make a cocktail in a bigger tube consisting of the buffer, 2 enzymes, and water. The rationale behind thissis that you'll be less likely to make a bigger mistake with a bigger volume (versus getting 1 microL of enzyme per specific DNA). You'll also know that there is no problem with the enzyme if all the DNA don't cut. If only one specific DNA doesn't work, then we'll know that it's because there's something wrong with the specific DNA rather than anything in the "Master Mix." Since we have to do 11 digestions, we'll make enough of the master mix for 12 tubes to make sure we have enough. We'll take 17 microL of the Master Mix to each tube of DNA.

The optimal temperature for most enzymes is 37 degreees celsius. You'd like to have enzymes with the same optimal temperatures (for ex. restriction enzymes that start with B usually have an optimal temperature near 50 degrees. You wouldn't want to use both B-enzyme and EcoR1 because EcoR1 would die).

'''What percent gel should we use?'''

Look at the Davidson lab protocol on the Davidson-Missouri Wiki. Generally, bigger molecules require an agarose gel with a lower concentration.

'''What's next in the future?'''

5' end additions are done by PCR. We'll ampliffy this double-stranded DNA. We'll cut with both EcoR1 and BamH1 or Nco1 (depending on which reporter gene you're using). Then we'll cut the plasmid with the reporter with the same restriction enzymes used.Then, to throw away the remaining DNA that we don't want, we'll run a gel and throw away the smaller band (because the larger band is probably made of the plasmid we want to keep).Then we'll take the double stranded DNA we amplified and the purified plasmid and ligate, transform, and screen them and verify the sequence.

While we wait for the oligos, we can cut the plasmid with reporter on the same restriction enzyme already.

'''How are we building the tRNAs?'''

We'll assemble the oligos into genes by mixing our oligos together, boiling them, and very slowly allowing them to cool. This will require many calculations. We'll cut the plasmids with EcoR1 and Pst1. to be ready to receive the enes we're assembling.We'll have to gel purify the plasmids to get them away from their inserts. We'll ligate, transform, and screen the genes and the plasmids and verify the sequence. On paper you shouldn't have two sites cut with different restriction enzymes stick together, but if there's selection pressure, these sites may cut some ends to fit together. This is why we must screen them.

After Dr. Campbell taught us all of the above, the 5 biologists put together a document outlining exactly how we would go about cutting the two sets of plasmids (one set will be cut with Eco R1 and Pst1 and othe other set will be cut with Eco R1 and BamH1 or Nco1). The following table outlines which biologist was going to work with which sample of DNA:

[[Image:Names and DNA.jpg]]

The following summarize the diferent materials and volumes of these materials that we'll need for our digestion of the plasmids:

[[Image:Digestion materials.png]]

This last table highlights the agarose percent concentration we should use for each part according to how many base pairs it contains:

[[Image:Screen-capture-3.png]]

The biologists digested the plasmids that we plan on using once our oligos arrive in a day or two. Afterwards, we made the two agarose gels that were most compatible with the parts we were going to run. This gel is going to purify the plasmids so that we have the parts that we're going to use later on, minus the rest of the DNA in the plasmid that we don't need. Before physically creating the gel, we figured out how much agarose we would need to make our two different agarose gel concentrations, 0.8% and 2.5%. Next, we made sure the mold and the big teeth were in place. We used the big teeth at the end of the mold because we plan on running 20 uL of DNA versus a running a smaller portion which would require us to use small teeth. We then measured the amount of agarose that we calculated we would need, placed this in a volumetric flask greater than 60 uL to allow for fizz to rise up, then measured out 60 uL of buffer and mixed this in the same flask with the agarose. We placed clear plastic wrap on top of the flask and heated it up for 1 minute and 20 seconds in the microwave. After we heated it, we took it out and checked to see if there was still agarose present that hadn't yet melted. There was, so we placed it back in the microwave and heated it for another 20 seconds. All the agarose had melted. We added 1 uL of EtBr (ethidium bromide) to the solution and quickly poured this into the mold. It needs about 15 to 20 minutes to cool. Dr. Campbell made the agarose gel that was 2.5% concentrated gel while we watched. We then made the 0.8% concentrated gel while he watched.

The following table lists the order in which we loaded our DNA into the gel. The first column all the way to the left is represented by number 1:

[[Image:Screen-capture-2.png]]

== [[User:Roclemente|Roclemente]] 08:46, 10 June 2009 (EDT) ==

After about 30 minutes after we began the gel electrophoresis, we looked at our gels with UV light to see how far the inserts had travelled and where the plasmids were. Then we took each gel to be photographed in the freezer room (231). We used the following DNA length marker to measure how long each sequence represented by each band was:

[[Image:Screen-capture-1.png]]

We compared our data to the lengths we expected to get according to the registry online. The first gel we photographed was the one that was 2.5% concentrated:

[[Image:2009-06-09 14hr 44min.jpg]]

The first two columns of the DNA we ran, both consisting of B0300, were fairly the same as the length we expected. However, the last two columns, consisting of K091111 and K091112 respectively were supposed to be 86 bp long, however, according to our data, K091112 was longer. Leland and I looked at Pallavi Penumetcha's paper which delved more closely into these two parts and what she wrote in her paper and what she entered into the registry conflict. We are currently corresponding with her to figure out the exact discrepancy.

We then photographed the 0.8% gel:

[[Image:2009-06-09 15hr 21min.jpg]]

The length of the sequences in almost every column matched up with the lengths we expected. Only the 4th and 5th columns didn't come out clearly. We think that the DNA inserted in column 5 weren't inserted properly and diffused on its own. We couldn't see bands in the 4th column at all until we adjusted the color a bit on the photographer and saw that column 4 was pretty much identical to column 5. We assumed that column 5 bled into column 4.

== [[User:Roclemente|Roclemente]] 15:14, 11 June 2009 (EDT) ==

We are digesting two parts today: I715039 (pLac+RBS+RFP) and S03710 (Tet Resistance). We want both the plasmids and the inserts in each case. We are aiming for a final volume of 40 uL for each part. This means that each digestion will consist of 4 uL buffer, 2 uL EcoR1, and 2 uL of their other respective restriction site (BamH1 for S03710 and Nco1 for I715039). The only parts that will vary are the amount of DNA and water we use. The amount of DNA we use depends on the concentration of the DNA we have (found through use of the NanoDrop). Since we want 2500 ng of DNA, we can use the concentration of DNA to figure out how much volume of this concentrated DNA we'll want to use in your digestion. If we subtract the volumes of buffer, two restriction enzymes, and DNA we plan on using from 40 uL, we'll have the amount of water we need to add. Here are the different volumes of materials we plan on using in our double digestion:

[[Image:DoubledigestRFPTetR.png]]

Now that we know how much of each material we were going to use, we needed to find out which buffer would be compatible with each sample according to the restriction enzymes we were using and how effective they'd be in different salt solutions. Online on the Promega "Compatible Buffers" website, we were able to find which Promega buffers would be most compatible when doing a double digest with EcoR1 and BamH1:

[[Image:BamEcoR1.png]]

Here is a chart noting buffers and their compatiblity with EcoR1 and Nco1:

[[Image:NcoEcoR1.png]]

We still have to wait for the restriction enzymes to arrive, so afterwards, we transformed three different inserts containing needed promoters and RBSs into vectors. We used the Zippy Transformation method. These promoters and RBSs will later be added to the FMLs once they are already inserted into the reporter cells. The following table outlines these inserts and part numbers:

[[Image:Promoterinsertspartnumbers.png]]

The restriction enzymes have arrived. We will now be able to do PCR to amplify the RFP gene (I715039) and the Tet resistance gene (S03710) as templates. We will be using the primers which have also arrived. We ordered forward and reverse primers so that our amplified sequence will consist of the following in this order:

GCAT-ATG-5mer-Reporter Gene

The following table lists the materials will be needed for this PCR technique which uses the "Green Promega master (Monster) Mix:"

[[Image:Greenpromegamastermix.png]]

This is the PCR procedure:

[[Image:Temperaturecycle.png]]

These are the variables that we will be adding to each separate sequence we want to be amplified:

[[Image:Tobeamplified.png]]

This table shows how we labeled the tubes based on how we divided the PCR work for the 10 tubes:

[[Image:Pcrtubes.png]]

Now we are giong to assemble the tRNA oligos together to form the tRNA genes. We could only order a certain length of sequence and the tRNA genes outdid these specified lengths. We used the Lancilator to find various oligonucleotides that overlapped and would be able to anneal together to form the tRNA genes. We used the Davidson lab protocol entitled "Buiding dsDNA with Oligos" to assemble the oligos:

[[Image:Oligoassembly.png]]

== [[User:Roclemente|Roclemente]] 22:46, 12 June 2009 (EDT) ==

We had two main goals for today:
1. perform a tRNA ligation and transformation
2. perform two electrophoreses: one to verify the PCR sequences we obtained and another to purify the digested reporter plasmids we would need

We began the day by pouring the agarose gels according to which concentration of buffer would be most compatible with the molecular weight of the substance we wanted to isolate. For both the digestion of E01010 and J31007, according to the "Optimize your Gel" website found on the Davidson Lab Protocols site for iGEM 2009, we wanted a 0.4% concentrated gel. For the PCRs, we wanted a 1.6% concentrated gel to optimize our retrieval of the Tet gene and a 1.4% concentrated gel to optimize our retrieval of the RFP gene.

While we waited for the gels to harden for about 30 minutes, we began calculating how much of each material we would need to ligate our tRNAs. We used the following equation to find out how much insert we should use:

[[Image:Ligationequation.png]]

Using the lengths of sequences we would be ligating, this is the mass that we needed:

X nanograms of insert = [(2)(144bp)(50 ng linearalized plasmid)]/2038bp
X = 7.0657 ng of insert

The following table outlines our calculations for finding the mass of tRNAs we had:

[[Image:Ligationcalculations.png]]

We would attain this mass of tRNA by pipetting an unknown amount of it. In order to attain a more "pipetable" amount of tRNA, we diluted our initial concentration 50X. Since we knew we wanted our final mass to be 7.065 ng, we calculated the volume we would need to take from this new, diluted sample:

DILUTION: 555.48/50 = 11.012
CALCULATION: 11.012 ng/ 1uL = 7.065 ng/ Y uL
Y = .64 uL of insert

Afterwards, Olivia and Alyndria began the actual ligation process while Shamita, Leland, and I filled the wells of the two gels. The first included the two digested reporter parts. We also included a molecular weight marker. Here is a photograph of this gel:

[[Image:2009-06-12 15hr 54min.jpg]]

According to our prior calculations, the Tet plasmid should have been 2958 base pairs long and the RFP plasmid should have been 2380 base pairs long. Our expectations were verified by the data. Shamita and I then sliced the part of thsi gel containing both plasmids and put these slices away to purfiy on Monday.

The second gel contained aliquots of the PCR we had done to attain 10 FML sequences: 5 containing the Tet resistant reporter gene and each of the 5 frameshifts that Davidson was assigned and the other 5 containing the RFP reporter gene and each of the 5 frameshifts as well:

[[Image:2009-06-12 16hr 07min.jpg]]

According to our data, each sequence containing the Tet resistant gene should have been 317 base pairs long and each sequence containing the RFP gene should have been 446 base pairs long. Our data supports our hypothesis, however, the 3rd well was not successful. This well contained the FML consisting of the Tet resistant gene and the frameshift mutant CUAGU. Before leaving work for the day, Shamita and I prepared and performed PCR using two parts containing the tet resistant gene, J31007 and S03710. We wanted to use both to make sure at least one of them works since we could technically use either of them, even though when we first did PCR we only used the S03710 part. Using the known concentrations of each of these parts, and knowing that we needed 1 ng of each part to include in our PCR samples, we calculated the volume of each 100X-diluted solution we would need:

S03710: 1.636 ng/uL = 1 ng/ X uL
X = 0.611 uL

J30017: 0.776 ng/uL = 1 ng/ Y uL
Y = 1.289 uL

We will run another gel with both of these parts on Monday and hopefully at least one of them works.

== [[User:Roclemente|Roclemente]] 17:24, 15 June 2009 (EDT) ==

Each biology member of the Davidson iGEM team has been assigned a 5mer to carry on throughout the rest of their respective processes. I've been assigned the 5mer CUAGU. We verified the PCR of the CUAGU FML containing the RFP gene on Friday. However, we weren't able to verify the CUAGU FML containing the tet resistance gene when we ran it on a gel. Shamita and I redid PCR for two parts containing the tet resistance gene: J31007 and S03710, labeled 2* and 2! respectively. I'm running another gel to verify either or both of them. Both sequences should be 317 bp long (this includes the ATF, logical clause, and the tet resistance gene to the left of the BamH1 restriction site). Here is the gel run we collected. The first column represents the 1 kb DNA marker, and the next two are 2* then 2!:

[[Image:2009-06-15 10hr 34min.jpg]]

The data shows that the PCRs of both parts wound up at the length we expected. The first time we did PCR on J31007, we must have left a solution out of our procedure on accident. I combined both PCRs together since they are the same length and match the length we expected to get.

Next, we cleaned and concentrated our amplified DNA sequence (#D4014 from Zymo Research). I cleaned the two pieces of DNA that contained the 5mer CUAGU. I labeled the tube with DNA also containing the RFP gene as "1" and the tube with DNA also containing the tet resistance gene as "2!". We needed 2 volumes of buffer for every 1 volume of DNA we added. Therefore, for tube "1," I added 95 uL DNA and 190 uL buffer. I added more DNA to tube "2!" since I combined the DNA from two PCR events to one (2* and 2! from earlier).

After cleaning the DNA, we found its concentration using the Nanodrop. In a solution made up of 0.5 uL of the DNA and 2.5 uL of TE, we found the following data from the DNA in this solution:

[[Image:Cleanconcentrate.png]]

The concentration of the DNA we cleaned was the concentration given by the Nanodrop multiplied by 6 (the DNA was dilute 6-fold for the Nanodrop). We used this concentration and the volume of clean DNA we knew we had (9.5 uL) to find the mass of clean DNA we had:

[[Image:Concentrateddna.png]]

Before doing digestion, we used the ligation equation (see "Ligation Protocol" in "Davidson Lab Protocols") to figure out how much mass of the DNA insert we would need later on when we did ligation. For the RFP FML, we found we would want the following mass of insert:

ng of insert = (2)(443)(50)/(2320) = 19.0948 ng

We multiplied this mass by 50 because we're counting on losing DNA between the digestion and cleaning we would need to do leading up to the ligation protocol.

19.0948 ng x 50 = 954.741 ng RFP FML

We found the mass of Tet FML insert we would need for ligation as well:

ng of insert = (2)(314)(50)/(2958) = 10.6153 ng

We multiplied this mass by 50 too:

10.6153 x 50 = 530.764 ng Tet FML

Unfortunately, nobody had enough of their clean DNA to satisfy our careful criteria. However, we would need to do this calculation later on so it was helpful we did it anyway. Instead, we just used all the DNA we had, or 9.5 uL for each reporter gene. Since there were 4 people who needed the same master mix, we made 5 parts of the master mix using the following volumes:

[[Image:Rfptetdigestion.png]]

After realizing that we would have to wait a day for the digestion to take place, we decided to culture every tRNA cell that colonized. The tRNA that we transformed on Friday did not have a good yield, so last night a few people came in and transformed more tRNA cells. We got a few more colonies. To be safe and make sure we cultured each tRNA, we picked every colony visible to us in both the old and new plates. Using our own given tRNAs, I counted the amount of colonies that grew in the plate we transformed on 6/12 and 6/14. There were 4 colonies that grew on both plates so I labeled a tube for each of these. I awas also in charge of the negative control. There were 2 colonies that grew on the negative control plate so I labeled 2 tubes for that. We will be placing them in an incubator overnight.

== [[User:Roclemente|Roclemente]] 12:30, 16 June 2009 (EDT) ==

We checked on the cells we cultured overnight. Of the cells that I cultured (all containing the CUAGU 5mer and the two negative controls), both negative control tubes worked as well as the tubes I labeled 1 (6/14), 5 (6/12), and 7 (6/12). The rest of mine were either red, meaning the plasmids hadn't ligated properly) or the colonies we used were no good. I took these 5 tubes, along with 3 of Olivia's tubes filled with cells that were cultured (tubes 11, 12, and 13) and miniprepped them to isolate the plasmids from the cells. There was no need to colony PCR to screen for successful ligations because this is only used to know which ones to let grow overnight and we had already done this.

As soon as I finished miniprepping them, I had to leave the lab for a little bit, so Olivia took each my miniprep tubes besides the negative controls and Leland took my negative controls. I completed the protocol for digestion of the tRNAs with EcoR1 and Pst1 for Alyndria's tubes when I got back because she had left a little earlier for lunch.

Afterwards, Shamita and I poured a 1.9% concentrated gel to load the digested tRNAs on. We want to verify that these tRNAs are the appropriate size for if they were cut properly. Since we will have to load 37 wells, and the maximum number we can load on one gel is 18 since 2 wells will have to be used by the molecular weight marker, we needed to pour 2 gels and we will also be using a half of a 2.0% agarose gel that was left over from another experiment.

Shamita and I also made new 0.5X running buffer gel. We did this by adding 450 uL buffer and 9 uL ethidium bromide (1 uL EtBr : 50 uL buffer). Alyndria, Leland, and Shamita loaded these gels.

Olivia and I cleaned our PCR DNA using the "Ethanol precipitate DNA (short protocol)" found in the Davidson Lab Protocols link. To begin with, I added 180 uL dH2O to my two samples, CUAGU with RFP and CUAGU with Tet, to bring their volumes up to 200 uL. During one of the last steps of this protocol, when Olivia and I dried out our DNA using the SpeedVac for a period of 10 minutes, the top of my tube containing the CUAGU 5mer and RFP gene broke. I wasn't sure if a pellet was in there, but I still poured out the ethanol and went on with the protocol. At the end, I resuspended what may or may not have been my DNA in 10 uL distilled water. I NanoDropped it afterward and attained the following data concerning both tubes:

[[Image:Failedpcrmeh.png]]

According to this data, any absorption and concentration we may have attained is insignificant because the A-260 must be between 0.05 and 1.50 to be significant and neither of the DNA is. Tomorrow I will do PCR for both FMLs over again.

== [[User:Roclemente|Roclemente]] 14:51, 17 June 2009 (EDT) ==

I found out that my tRNA DNA that I labeled "1" and which contains CUAGU was verified from the gel run Olivia did yesterday (the gel run also included her tRNA DNA samples). At the start of the day I plated this tube of cultured cells again to grow overnight.

Since PCR will take about 3 1/2 hours, I attempted this procedure at the beginning of the day. I plan on making 5 PCR products for each reporter gene since I now know the environmental conditions are correct. I labeled the RFP tubes 1-5 and the Tet tubes 6-10. I first needed to figure out how much of the template DNA I would need in order to have 1 ng template DNA. I made the following calculations to find this out:

[[Image:1ngtemplatedna.png]]

I wanted to add the Monster Mix last, so I made the following table, adding everything else besides the Monster Mix for 5 of the same reporter gene to one tube:

[[Image:Pcrmix.png]]

Unfortunately, when I actually mixed the solution together, I forgot to dilute the template DNA. Therefore, we have 100X more template DNA than we would have had we diluted the template DNA. The PCR can still work, but we will have to constantly remind ourselves that there will be extra template DNA in our solution and that this may skew, however slightly, our results later on.

While I waited for the PCR product, I refocused on getting my tRNA DNA ready for sequencing. Using the NanoDrop, I found the concentration of my miniprepped CUAGU DNA that was verified to be 61 ng/uL. The pre-sequencing protocol calls for 60 ng DNA, so I extracted 1 uL from this tube (the extra DNA will be insignificant in the sequencing procedure). I pipetted this into another tube and used the SpeedVac to dry it out. This is how we will send our DNA to be sequenced.

The following is a summary of steps I put together to help me plan out what I will do after the PCR procedure is over (it will be done at 5 pm today):

1. Combine the five matching tubes into one tube.
2. Run each PCR product on a gel to verify that our expected sequence has been properly amplified.
3. Clean and concentrate plasmids so salt solution is optimal for digestion.
4. Double digest PCR products with EcoR1 and BamH1 or Nco1.
5. Clean and concentrate again to get rid of restriction enzymes.
6. Nanodrop inserts in order to do ligation calculations and verify that sequences are present.
7. Ligate FML inserts with purified plasmids.
8. Transform plasmid into cells.

Since I won't have time to run a gel and take a picture once the PCR procedure is complete (we get out at 5:30 and it will be finished at 5:00), I have begun to prepare the agarose gel for use tomorrow. The size of the band I expect to find for the Tet FML is 349 bp (including the GCAT, BioBrick prefix, ATG, 5mer, and reporter gene until about 20 nucleotides or so past the restriction site we will cut at) and 474 bp for the RFP FML. According to the "Optimize Your Gel" link on the Davidson Lab Protocols page, a 1.6% gel will optimize how well I see my Tet bands and a 1.3% gel will optimize how well I see my RFP bands. I can use a gel with a concentration in between both of the optimal concentrations for Tet and RFP so that I can use one gel for both. Therefore, I will use a 1.45% gel. 1.45% of 60 uL buffer solution tells me that I should measure out 0.87 g agar for this gel.

== [[User:Roclemente|Roclemente]] 14:00, 18 June 2009 (EDT) ==

I checked on my PCR products this morning. Tubes 1 and 2 (both containing the RFP FML) had solution scattered on the tube's cover and walls so I placed them in the centrifuge for a bit. Unfortunately, I forgot to put these 500 uL tubes in holders when I centrifuged them so these tubes were destroyed. This was okay though because I was just going to combine each respective FML into one tube. I would have 300 uL of RFP FML PCR product instead of 500 uL and I put all of this in tube 5. I still had 500 uL of Tet FML PCR product left and put all of this in tube 10.

I ran 5 uL of both RFP and Tet FMLs on a gel, as well as a 1 kb molecular weight marker. The MW marker is in lane 1, the RFP is in lane 3, and the Tet is in lane 4 (Leland ran his PCR product in lane 2):

[[Image:2009-06-18 09hr 56min.jpg|thumb|none|100px]]

I was expecting the RFP PCR product to be 474 bp long and the Tet PCR product to be 349 bp long. Both appear to come out a bit shorter, but Dr. Campbell and I agreed that we wouldn't count these sequences out because the bands were quite close to what we expected.

I proceeded to clean and concentrate these two sequences using the procedure outlined in the "Clean and Concentrate DNA (after PCR, before digestion)." Now I had 295 uL of the Tet sequence and 495 uL of the RFP sequence. Since the procedure says I can only have 800 uL in the column at a time, with DNA:Buffer at a ratio of 1:2, I mixed the two in a column in a series of two rounds using the following volumes:

[[Image:Clean.png]]

I Nanodropped my DNA after cleaning it and attained the following results:

[[Image:Nanome.png]]

I wanted the OD260 (Optical Density at 260) to be under 1.5, which it is for both. I lost quite a bit of Tet DNA in the process, and will only continue to lose DNA. To account for this loss of DNA, Dr. Campbell suggested it would be a good idea to heat inactivate the restriction enzymes to get rid of them after digestion instead of cleaning and concentrating the DNA over again.

I double digested both RFP and Tet FML sequences. I used all 9.5 uL of my DNA. The following table outlines how much volume of each material I mixed together:

[[Image:2nddoubledigest.png]]

While I waited 3 hours for my digestion to take place, I prepared 6 tubes to make more liquid cultures of the RFP and Tet vectors to grow more receiving vectors. 3 tubes would be to make RFP cultures and the other 3 would be for the Tet cultures. For RFP, I used the part I715039 and for Tet, I used the part J31007.

Once the digestion took place, Dr. Campbell notified me that heat inactivation was capable for Nco1 but not BamH1. There were 3 options for me to take in order to clean the Tet FML, the protocols for all of which can be found on the Davidson Lab Protocols link:

# [http://www.bio.davidson.edu/courses/Molbio/Protocols/Clean_Concentrate.html Clean and Concentrate DNA (after PCR, before digestion)]
# [http://www.bio.davidson.edu/courses/Molbio/Protocols/clean_short.html Ethanol Precipitate DNA (short protocol)]
# [http://www.bio.davidson.edu/courses/Molbio/Protocols/Qiagen_gelpure.html Qiagen QIAquick Gel Purification]

Although I could've chosen to do the heat inactivation for the RFP FML, I chose to keep the purifying technique in synch for consistency's sake. I chose to do the gel purification. While this process takes longer than the other two processes, I have found that gel purification generates the greatest yield and therefore chose it.

IGEM 2009 notebook

2009-06-18T20:40:39Z

ShPunjabi: /* Roclemente 14:00, 18 June 2009 (EDT) */

== [[User:Roclemente|Roclemente]] 16:54, 3 June 2009 (EDT) ==

Shamita and I are trying to find suitable reporter proteins to use. Yesterday, Leland and Alyndria were working on ways to insert the gene sequences into the plasmid. Upon seeing how they wanted to manipulated the reporter gene to include the logical clauses, we came up with a few criteria for the reporter genes we would use. The following criteria for genes are listed in order of the broadest aspect to look at to the narrowest aspect:

a) Doesn't contain restriction sites for the 4 restriction enzymes (EcoR1, Xbal, Spel, Pst1) used to cleave the Biobrick part out of the plasmid.

b) Contains 6 cutter restriction sites.

c) These restriction enzymes aren't blunt (cleave straight down at one spot).

d) These restriction sites are close to thge 5' (beginning) end of the sequence.

e) These enzymes are easiest to work with and cheapest.

We are finding the part numbers of the reporter genes we want to use (antibiotic resistance, fluorescence, ''LacZ'') through our own GCAT because we know these ones work. We are then locating these parts on the parts registry [http://www.partsregistry.org] website. We copied and pasted the gene sequences we obtained from the registry onto the ApE software [http://www.biology.utah.edu/jorgensen/wayned/ape/]. From here, we were able to generate a genetic map of each gene that outlined each restriction site that fit our criteria. We put each genetic map, alongside the part number used, into a Word document:

http://gcat.davidson.edu/GcatWiki/images/0/0e/Restriction_Site_Mapping_on_Reporter_Genes.doc

So it looks like we've changed directions. The team is looking towards the wet lab portion of our research more now. Instead of simply speculating about which reporter proteins we think will work best and which tRNA suppressors to use, we're going to physically test it out ourselves. A few tasks at hand in the second part of this afternoon:

1. What are the DNA gene sequences of the 12 different tRNA suppressors we want to use?

2. What is the sequence of the DNA that is cleaved off of both ends of the tRNA when it is transcribed?

We e-mailed Anderson to find out the exact tRNA gene sequences because we had a hard time finding this information everywhere else. Most papers we found describing the 5-base codon tRNA suppressors simply referred to Anderson's paper [http://gcat.davidson.edu/GcatWiki/images/6/6f/AndersonSchultz2002ChemBiol.pdf]. Anderson replied near the end of the day with the gene sequence. According to his email, the only difference between different tRNA genes is the anticodon loop. Therefore, if we just substitute all the anticodon loops in the invariable part of the sequence, we have our different tRNA sequences. We plan on using the Lancilator to help us break these tRNA sequences into smaller oligonucleotides that can anneal at around the same temperature because it is not possible for us to order oligos that are greater than 70 nucleotides long.

== [[User:Roclemente|Roclemente]] 17:21, 4 June 2009 (EDT) ==

I started out the day looking at my time-sensitive group research qustion about reporter proteins. I started making a table listing the advantages and disadvantages of different reporter proteins. Then the group found out that there was a major roadblock to our project: a stop codon was located in our ATG-5mer oligo. The stop codon would have been part of the BioBrick scar. The group looked at several ways to work around this problem. The idea that I looked most into was replacing the restriction sites that were used in the scar through standard assembly. According to the judging criteria for iGEM, I found that a team could possibly alter the standard assembly method as long as they wrote a letter to the iGEM judges explaining our plan in a detailed manner. From there, I set off to understand the BioBrick scar more by making a document highlighting the exact positions of the rsetriction sites on the BioBrick parts and how they were used to put more than one part together. I then found several other pairs of restriction sites that complemented one another and could be used in place of the standard scar between Xba1 and Spe1. At the end of the day, the group got the chance to speak with Dr. Campbell and he suggested a hybrid idea which would combine Davidson's restriction site idea with Missouri's PCR idea. I'm not quite sure of what this idea is exactly yet, but we'll all clarify our vague idea of it tomorrow morning after a talk with Dr. Eckdahl.

== [[User:Roclemente|Roclemente]] 17:13, 5 June 2009 (EDT) ==

This morning we began exploring different options for bypassing the stop codon problem from yesterday. If we wanted to make sure we were using the BioBrick parts in our GCatalog, there was no way we were going to be getting rid of the Xba1 restriction site that contained the "TAG" which codes for a stop codon. Therefore, we proposed looking at restriction sites within the reporter genes instead, therefore addressing the already-placed BioBrick ends. Using the document me and Shamita created on "Restriction Site Mapping on Reporter Genes" (see first journal entry), we determined that the reporter gene with the restriction site closest to the beginning of the sequence was YFP. We proposed cutting the YFP gene that we already had in stock at this restriction site and inserting an oligo (which we would have ordered) with a sequence in the following order: "BioBrick prefix - ATG - 5mer - part of YFP gene to the left of the cleaved restriction site."

After clarifying our own idea with one another, we spoke with some Missouri kids to clarify the said "hybrid" idea which, according to Dr. Campbell, combined the ideas of both campuses. The hybrid idea was based upon our idea of cutting the reporter gene at a restriction site as well, except that our primer would start out with a primer for our reporter gene, and to the left of the primer would be a "tail" consisting of the ATG and 5mer and either an Xba1 or EcoR1 restriction site (We still have to decide this. Our decision will probably be based on the restriction site we end up using in our reporter gene and whether or not that is complementary to either the Xba1 or EcoR1 sites.). We would use the PCR technique and use our in-stock reporter gene strand as our template strand (it will be denatured into single strands) and we will add our oligo. We propose that the primer will synthesize the rest of the YFP strand as well as add on nucleotides to complete the oligo tail. In the end, we will have a double stranded DNA sequence consisting of either the Xba1 or EcoR1 site, the ATG, the 5mer, and the reporter gene. The beauty of this hybrid idea is that we have flexibility as far as the reporter gene we choose to use since all we really need for it is the beginning sequence (for the primer) and a known, "hearty" restriction site. Once we cut this double strand at this restriction site on the reporter gene as well as at the Xba1 or EcoR1 sites, we can insert this into a plasmid that contains the rest of that reporter gene after the restriction site we choose to use and the complementary Xba1 or EcoR1 site.

I made a Word document outlining the promoter and RBS parts we have in our registry on the Davidson campus. Leland and I went through the registry parts on the GCATalog to look for potential promoters and RBS parts. We went through the document as a group and analyzed each of these potential parts on partsregistry.org to see which ones would most likely work the best in the miniprep we'll start preparing for on Sunday evening. Here is the document we wound up with:

http://gcat.davidson.edu/GcatWiki/images/d/d2/Promoters_and_RBS.doc

== [[User:Roclemente|Roclemente]] 10:40, 8 June 2009 (EDT) ==

We're starting the day by finalizing the oligos we will be ordering to have synthesized. Two groups of oligos must be sequenced: primers and tRNA gene sequence. The primers will consist of a forward and reverse primer. The forward primer will consist of the following in this 5' - 3' order: GCAT - BioBrick Prefix - ATF - 5mer - first 20 nucleotides in the reporter gene sequence. These 20 nucleotides won't include the ATG that naturally comes at the beginning of the reporter gene because we want to make sure the ribosome attaches to the first ATG and reads the logical clause instead of going straight to the ATG at the start of the reporter gene. The reverse primer will consist of the first 20 nucleotides downstream of the restriction site we plan to use in the reporter gene. I worked on finding the primers for the Tet resistance gene (part J31007) while Lyn worked on finding the primers for the RGP gene. Dr. Campbell decided we should use the BamH1 restriction enzyme because it is very "hardy." The following document outlines the 5 forward primers (each with one of the five variable 5mers) we will order for amplification of the Tet resistance FML:

[[Image:Tet resistance forward primers.png]]

This is the reverse primer we will order for the Tet resistance FML:

[[Image:Tet resistsance reverse primer.png]]

== [[User:Roclemente|Roclemente]] 11:54, 9 June 2009 (EDT) ==

We need to digest the plasmids at the EcoR1 and Pst siets. Everytime we're going to set up a digestion, we'll want the following in these volumes: 10x Buffer (2 uL), DNA (~ 3 uL), enzymes 1 and 2 (1 uL each), water (13 uL), and final volume of all of these put together should be 20 uL.

Some notes about digestion: Enzymes should make up 10% of the final volume. As a general rule of thumb, make sure the tip of your pipet is close to the surface of the solution. This is especially pertinent when gathering 1 microL of each of the enzymes. The most sensitive components are the enzymes, so they should go last.

We'll have a bunch of tubes with each 3 microL of the specific DNA. We'll make a cocktail in a bigger tube consisting of the buffer, 2 enzymes, and water. The rationale behind thissis that you'll be less likely to make a bigger mistake with a bigger volume (versus getting 1 microL of enzyme per specific DNA). You'll also know that there is no problem with the enzyme if all the DNA don't cut. If only one specific DNA doesn't work, then we'll know that it's because there's something wrong with the specific DNA rather than anything in the "Master Mix." Since we have to do 11 digestions, we'll make enough of the master mix for 12 tubes to make sure we have enough. We'll take 17 microL of the Master Mix to each tube of DNA.

The optimal temperature for most enzymes is 37 degreees celsius. You'd like to have enzymes with the same optimal temperatures (for ex. restriction enzymes that start with B usually have an optimal temperature near 50 degrees. You wouldn't want to use both B-enzyme and EcoR1 because EcoR1 would die).

'''What percent gel should we use?'''

Look at the Davidson lab protocol on the Davidson-Missouri Wiki. Generally, bigger molecules require an agarose gel with a lower concentration.

'''What's next in the future?'''

5' end additions are done by PCR. We'll ampliffy this double-stranded DNA. We'll cut with both EcoR1 and BamH1 or Nco1 (depending on which reporter gene you're using). Then we'll cut the plasmid with the reporter with the same restriction enzymes used.Then, to throw away the remaining DNA that we don't want, we'll run a gel and throw away the smaller band (because the larger band is probably made of the plasmid we want to keep).Then we'll take the double stranded DNA we amplified and the purified plasmid and ligate, transform, and screen them and verify the sequence.

While we wait for the oligos, we can cut the plasmid with reporter on the same restriction enzyme already.

'''How are we building the tRNAs?'''

We'll assemble the oligos into genes by mixing our oligos together, boiling them, and very slowly allowing them to cool. This will require many calculations. We'll cut the plasmids with EcoR1 and Pst1. to be ready to receive the enes we're assembling.We'll have to gel purify the plasmids to get them away from their inserts. We'll ligate, transform, and screen the genes and the plasmids and verify the sequence. On paper you shouldn't have two sites cut with different restriction enzymes stick together, but if there's selection pressure, these sites may cut some ends to fit together. This is why we must screen them.

After Dr. Campbell taught us all of the above, the 5 biologists put together a document outlining exactly how we would go about cutting the two sets of plasmids (one set will be cut with Eco R1 and Pst1 and othe other set will be cut with Eco R1 and BamH1 or Nco1). The following table outlines which biologist was going to work with which sample of DNA:

[[Image:Names and DNA.jpg]]

The following summarize the diferent materials and volumes of these materials that we'll need for our digestion of the plasmids:

[[Image:Digestion materials.png]]

This last table highlights the agarose percent concentration we should use for each part according to how many base pairs it contains:

[[Image:Screen-capture-3.png]]

The biologists digested the plasmids that we plan on using once our oligos arrive in a day or two. Afterwards, we made the two agarose gels that were most compatible with the parts we were going to run. This gel is going to purify the plasmids so that we have the parts that we're going to use later on, minus the rest of the DNA in the plasmid that we don't need. Before physically creating the gel, we figured out how much agarose we would need to make our two different agarose gel concentrations, 0.8% and 2.5%. Next, we made sure the mold and the big teeth were in place. We used the big teeth at the end of the mold because we plan on running 20 uL of DNA versus a running a smaller portion which would require us to use small teeth. We then measured the amount of agarose that we calculated we would need, placed this in a volumetric flask greater than 60 uL to allow for fizz to rise up, then measured out 60 uL of buffer and mixed this in the same flask with the agarose. We placed clear plastic wrap on top of the flask and heated it up for 1 minute and 20 seconds in the microwave. After we heated it, we took it out and checked to see if there was still agarose present that hadn't yet melted. There was, so we placed it back in the microwave and heated it for another 20 seconds. All the agarose had melted. We added 1 uL of EtBr (ethidium bromide) to the solution and quickly poured this into the mold. It needs about 15 to 20 minutes to cool. Dr. Campbell made the agarose gel that was 2.5% concentrated gel while we watched. We then made the 0.8% concentrated gel while he watched.

The following table lists the order in which we loaded our DNA into the gel. The first column all the way to the left is represented by number 1:

[[Image:Screen-capture-2.png]]

== [[User:Roclemente|Roclemente]] 08:46, 10 June 2009 (EDT) ==

After about 30 minutes after we began the gel electrophoresis, we looked at our gels with UV light to see how far the inserts had travelled and where the plasmids were. Then we took each gel to be photographed in the freezer room (231). We used the following DNA length marker to measure how long each sequence represented by each band was:

[[Image:Screen-capture-1.png]]

We compared our data to the lengths we expected to get according to the registry online. The first gel we photographed was the one that was 2.5% concentrated:

[[Image:2009-06-09 14hr 44min.jpg]]

The first two columns of the DNA we ran, both consisting of B0300, were fairly the same as the length we expected. However, the last two columns, consisting of K091111 and K091112 respectively were supposed to be 86 bp long, however, according to our data, K091112 was longer. Leland and I looked at Pallavi Penumetcha's paper which delved more closely into these two parts and what she wrote in her paper and what she entered into the registry conflict. We are currently corresponding with her to figure out the exact discrepancy.

We then photographed the 0.8% gel:

[[Image:2009-06-09 15hr 21min.jpg]]

The length of the sequences in almost every column matched up with the lengths we expected. Only the 4th and 5th columns didn't come out clearly. We think that the DNA inserted in column 5 weren't inserted properly and diffused on its own. We couldn't see bands in the 4th column at all until we adjusted the color a bit on the photographer and saw that column 4 was pretty much identical to column 5. We assumed that column 5 bled into column 4.

== [[User:Roclemente|Roclemente]] 15:14, 11 June 2009 (EDT) ==

We are digesting two parts today: I715039 (pLac+RBS+RFP) and S03710 (Tet Resistance). We want both the plasmids and the inserts in each case. We are aiming for a final volume of 40 uL for each part. This means that each digestion will consist of 4 uL buffer, 2 uL EcoR1, and 2 uL of their other respective restriction site (BamH1 for S03710 and Nco1 for I715039). The only parts that will vary are the amount of DNA and water we use. The amount of DNA we use depends on the concentration of the DNA we have (found through use of the NanoDrop). Since we want 2500 ng of DNA, we can use the concentration of DNA to figure out how much volume of this concentrated DNA we'll want to use in your digestion. If we subtract the volumes of buffer, two restriction enzymes, and DNA we plan on using from 40 uL, we'll have the amount of water we need to add. Here are the different volumes of materials we plan on using in our double digestion:

[[Image:DoubledigestRFPTetR.png]]

Now that we know how much of each material we were going to use, we needed to find out which buffer would be compatible with each sample according to the restriction enzymes we were using and how effective they'd be in different salt solutions. Online on the Promega "Compatible Buffers" website, we were able to find which Promega buffers would be most compatible when doing a double digest with EcoR1 and BamH1:

[[Image:BamEcoR1.png]]

Here is a chart noting buffers and their compatiblity with EcoR1 and Nco1:

[[Image:NcoEcoR1.png]]

We still have to wait for the restriction enzymes to arrive, so afterwards, we transformed three different inserts containing needed promoters and RBSs into vectors. We used the Zippy Transformation method. These promoters and RBSs will later be added to the FMLs once they are already inserted into the reporter cells. The following table outlines these inserts and part numbers:

[[Image:Promoterinsertspartnumbers.png]]

The restriction enzymes have arrived. We will now be able to do PCR to amplify the RFP gene (I715039) and the Tet resistance gene (S03710) as templates. We will be using the primers which have also arrived. We ordered forward and reverse primers so that our amplified sequence will consist of the following in this order:

GCAT-ATG-5mer-Reporter Gene

The following table lists the materials will be needed for this PCR technique which uses the "Green Promega master (Monster) Mix:"

[[Image:Greenpromegamastermix.png]]

This is the PCR procedure:

[[Image:Temperaturecycle.png]]

These are the variables that we will be adding to each separate sequence we want to be amplified:

[[Image:Tobeamplified.png]]

This table shows how we labeled the tubes based on how we divided the PCR work for the 10 tubes:

[[Image:Pcrtubes.png]]

Now we are giong to assemble the tRNA oligos together to form the tRNA genes. We could only order a certain length of sequence and the tRNA genes outdid these specified lengths. We used the Lancilator to find various oligonucleotides that overlapped and would be able to anneal together to form the tRNA genes. We used the Davidson lab protocol entitled "Buiding dsDNA with Oligos" to assemble the oligos:

[[Image:Oligoassembly.png]]

== [[User:Roclemente|Roclemente]] 22:46, 12 June 2009 (EDT) ==

We had two main goals for today:
1. perform a tRNA ligation and transformation
2. perform two electrophoreses: one to verify the PCR sequences we obtained and another to purify the digested reporter plasmids we would need

We began the day by pouring the agarose gels according to which concentration of buffer would be most compatible with the molecular weight of the substance we wanted to isolate. For both the digestion of E01010 and J31007, according to the "Optimize your Gel" website found on the Davidson Lab Protocols site for iGEM 2009, we wanted a 0.4% concentrated gel. For the PCRs, we wanted a 1.6% concentrated gel to optimize our retrieval of the Tet gene and a 1.4% concentrated gel to optimize our retrieval of the RFP gene.

While we waited for the gels to harden for about 30 minutes, we began calculating how much of each material we would need to ligate our tRNAs. We used the following equation to find out how much insert we should use:

[[Image:Ligationequation.png]]

Using the lengths of sequences we would be ligating, this is the mass that we needed:

X nanograms of insert = [(2)(144bp)(50 ng linearalized plasmid)]/2038bp
X = 7.0657 ng of insert

The following table outlines our calculations for finding the mass of tRNAs we had:

[[Image:Ligationcalculations.png]]

We would attain this mass of tRNA by pipetting an unknown amount of it. In order to attain a more "pipetable" amount of tRNA, we diluted our initial concentration 50X. Since we knew we wanted our final mass to be 7.065 ng, we calculated the volume we would need to take from this new, diluted sample:

DILUTION: 555.48/50 = 11.012
CALCULATION: 11.012 ng/ 1uL = 7.065 ng/ Y uL
Y = .64 uL of insert

Afterwards, Olivia and Alyndria began the actual ligation process while Shamita, Leland, and I filled the wells of the two gels. The first included the two digested reporter parts. We also included a molecular weight marker. Here is a photograph of this gel:

[[Image:2009-06-12 15hr 54min.jpg]]

According to our prior calculations, the Tet plasmid should have been 2958 base pairs long and the RFP plasmid should have been 2380 base pairs long. Our expectations were verified by the data. Shamita and I then sliced the part of thsi gel containing both plasmids and put these slices away to purfiy on Monday.

The second gel contained aliquots of the PCR we had done to attain 10 FML sequences: 5 containing the Tet resistant reporter gene and each of the 5 frameshifts that Davidson was assigned and the other 5 containing the RFP reporter gene and each of the 5 frameshifts as well:

[[Image:2009-06-12 16hr 07min.jpg]]

According to our data, each sequence containing the Tet resistant gene should have been 317 base pairs long and each sequence containing the RFP gene should have been 446 base pairs long. Our data supports our hypothesis, however, the 3rd well was not successful. This well contained the FML consisting of the Tet resistant gene and the frameshift mutant CUAGU. Before leaving work for the day, Shamita and I prepared and performed PCR using two parts containing the tet resistant gene, J31007 and S03710. We wanted to use both to make sure at least one of them works since we could technically use either of them, even though when we first did PCR we only used the S03710 part. Using the known concentrations of each of these parts, and knowing that we needed 1 ng of each part to include in our PCR samples, we calculated the volume of each 100X-diluted solution we would need:

S03710: 1.636 ng/uL = 1 ng/ X uL
X = 0.611 uL

J30017: 0.776 ng/uL = 1 ng/ Y uL
Y = 1.289 uL

We will run another gel with both of these parts on Monday and hopefully at least one of them works.

== [[User:Roclemente|Roclemente]] 17:24, 15 June 2009 (EDT) ==

Each biology member of the Davidson iGEM team has been assigned a 5mer to carry on throughout the rest of their respective processes. I've been assigned the 5mer CUAGU. We verified the PCR of the CUAGU FML containing the RFP gene on Friday. However, we weren't able to verify the CUAGU FML containing the tet resistance gene when we ran it on a gel. Shamita and I redid PCR for two parts containing the tet resistance gene: J31007 and S03710, labeled 2* and 2! respectively. I'm running another gel to verify either or both of them. Both sequences should be 317 bp long (this includes the ATF, logical clause, and the tet resistance gene to the left of the BamH1 restriction site). Here is the gel run we collected. The first column represents the 1 kb DNA marker, and the next two are 2* then 2!:

[[Image:2009-06-15 10hr 34min.jpg]]

The data shows that the PCRs of both parts wound up at the length we expected. The first time we did PCR on J31007, we must have left a solution out of our procedure on accident. I combined both PCRs together since they are the same length and match the length we expected to get.

Next, we cleaned and concentrated our amplified DNA sequence (#D4014 from Zymo Research). I cleaned the two pieces of DNA that contained the 5mer CUAGU. I labeled the tube with DNA also containing the RFP gene as "1" and the tube with DNA also containing the tet resistance gene as "2!". We needed 2 volumes of buffer for every 1 volume of DNA we added. Therefore, for tube "1," I added 95 uL DNA and 190 uL buffer. I added more DNA to tube "2!" since I combined the DNA from two PCR events to one (2* and 2! from earlier).

After cleaning the DNA, we found its concentration using the Nanodrop. In a solution made up of 0.5 uL of the DNA and 2.5 uL of TE, we found the following data from the DNA in this solution:

[[Image:Cleanconcentrate.png]]

The concentration of the DNA we cleaned was the concentration given by the Nanodrop multiplied by 6 (the DNA was dilute 6-fold for the Nanodrop). We used this concentration and the volume of clean DNA we knew we had (9.5 uL) to find the mass of clean DNA we had:

[[Image:Concentrateddna.png]]

Before doing digestion, we used the ligation equation (see "Ligation Protocol" in "Davidson Lab Protocols") to figure out how much mass of the DNA insert we would need later on when we did ligation. For the RFP FML, we found we would want the following mass of insert:

ng of insert = (2)(443)(50)/(2320) = 19.0948 ng

We multiplied this mass by 50 because we're counting on losing DNA between the digestion and cleaning we would need to do leading up to the ligation protocol.

19.0948 ng x 50 = 954.741 ng RFP FML

We found the mass of Tet FML insert we would need for ligation as well:

ng of insert = (2)(314)(50)/(2958) = 10.6153 ng

We multiplied this mass by 50 too:

10.6153 x 50 = 530.764 ng Tet FML

Unfortunately, nobody had enough of their clean DNA to satisfy our careful criteria. However, we would need to do this calculation later on so it was helpful we did it anyway. Instead, we just used all the DNA we had, or 9.5 uL for each reporter gene. Since there were 4 people who needed the same master mix, we made 5 parts of the master mix using the following volumes:

[[Image:Rfptetdigestion.png]]

After realizing that we would have to wait a day for the digestion to take place, we decided to culture every tRNA cell that colonized. The tRNA that we transformed on Friday did not have a good yield, so last night a few people came in and transformed more tRNA cells. We got a few more colonies. To be safe and make sure we cultured each tRNA, we picked every colony visible to us in both the old and new plates. Using our own given tRNAs, I counted the amount of colonies that grew in the plate we transformed on 6/12 and 6/14. There were 4 colonies that grew on both plates so I labeled a tube for each of these. I awas also in charge of the negative control. There were 2 colonies that grew on the negative control plate so I labeled 2 tubes for that. We will be placing them in an incubator overnight.

== [[User:Roclemente|Roclemente]] 12:30, 16 June 2009 (EDT) ==

We checked on the cells we cultured overnight. Of the cells that I cultured (all containing the CUAGU 5mer and the two negative controls), both negative control tubes worked as well as the tubes I labeled 1 (6/14), 5 (6/12), and 7 (6/12). The rest of mine were either red, meaning the plasmids hadn't ligated properly) or the colonies we used were no good. I took these 5 tubes, along with 3 of Olivia's tubes filled with cells that were cultured (tubes 11, 12, and 13) and miniprepped them to isolate the plasmids from the cells. There was no need to colony PCR to screen for successful ligations because this is only used to know which ones to let grow overnight and we had already done this.

As soon as I finished miniprepping them, I had to leave the lab for a little bit, so Olivia took each my miniprep tubes besides the negative controls and Leland took my negative controls. I completed the protocol for digestion of the tRNAs with EcoR1 and Pst1 for Alyndria's tubes when I got back because she had left a little earlier for lunch.

Afterwards, Shamita and I poured a 1.9% concentrated gel to load the digested tRNAs on. We want to verify that these tRNAs are the appropriate size for if they were cut properly. Since we will have to load 37 wells, and the maximum number we can load on one gel is 18 since 2 wells will have to be used by the molecular weight marker, we needed to pour 2 gels and we will also be using a half of a 2.0% agarose gel that was left over from another experiment.

Shamita and I also made new 0.5X running buffer gel. We did this by adding 450 uL buffer and 9 uL ethidium bromide (1 uL EtBr : 50 uL buffer). Alyndria, Leland, and Shamita loaded these gels.

Olivia and I cleaned our PCR DNA using the "Ethanol precipitate DNA (short protocol)" found in the Davidson Lab Protocols link. To begin with, I added 180 uL dH2O to my two samples, CUAGU with RFP and CUAGU with Tet, to bring their volumes up to 200 uL. During one of the last steps of this protocol, when Olivia and I dried out our DNA using the SpeedVac for a period of 10 minutes, the top of my tube containing the CUAGU 5mer and RFP gene broke. I wasn't sure if a pellet was in there, but I still poured out the ethanol and went on with the protocol. At the end, I resuspended what may or may not have been my DNA in 10 uL distilled water. I NanoDropped it afterward and attained the following data concerning both tubes:

[[Image:Failedpcrmeh.png]]

According to this data, any absorption and concentration we may have attained is insignificant because the A-260 must be between 0.05 and 1.50 to be significant and neither of the DNA is. Tomorrow I will do PCR for both FMLs over again.

== [[User:Roclemente|Roclemente]] 14:51, 17 June 2009 (EDT) ==

I found out that my tRNA DNA that I labeled "1" and which contains CUAGU was verified from the gel run Olivia did yesterday (the gel run also included her tRNA DNA samples). At the start of the day I plated this tube of cultured cells again to grow overnight.

Since PCR will take about 3 1/2 hours, I attempted this procedure at the beginning of the day. I plan on making 5 PCR products for each reporter gene since I now know the environmental conditions are correct. I labeled the RFP tubes 1-5 and the Tet tubes 6-10. I first needed to figure out how much of the template DNA I would need in order to have 1 ng template DNA. I made the following calculations to find this out:

[[Image:1ngtemplatedna.png]]

I wanted to add the Monster Mix last, so I made the following table, adding everything else besides the Monster Mix for 5 of the same reporter gene to one tube:

[[Image:Pcrmix.png]]

Unfortunately, when I actually mixed the solution together, I forgot to dilute the template DNA. Therefore, we have 100X more template DNA than we would have had we diluted the template DNA. The PCR can still work, but we will have to constantly remind ourselves that there will be extra template DNA in our solution and that this may skew, however slightly, our results later on.

While I waited for the PCR product, I refocused on getting my tRNA DNA ready for sequencing. Using the NanoDrop, I found the concentration of my miniprepped CUAGU DNA that was verified to be 61 ng/uL. The pre-sequencing protocol calls for 60 ng DNA, so I extracted 1 uL from this tube (the extra DNA will be insignificant in the sequencing procedure). I pipetted this into another tube and used the SpeedVac to dry it out. This is how we will send our DNA to be sequenced.

The following is a summary of steps I put together to help me plan out what I will do after the PCR procedure is over (it will be done at 5 pm today):

1. Combine the five matching tubes into one tube.
2. Run each PCR product on a gel to verify that our expected sequence has been properly amplified.
3. Clean and concentrate plasmids so salt solution is optimal for digestion.
4. Double digest PCR products with EcoR1 and BamH1 or Nco1.
5. Clean and concentrate again to get rid of restriction enzymes.
6. Nanodrop inserts in order to do ligation calculations and verify that sequences are present.
7. Ligate FML inserts with purified plasmids.
8. Transform plasmid into cells.

Since I won't have time to run a gel and take a picture once the PCR procedure is complete (we get out at 5:30 and it will be finished at 5:00), I have begun to prepare the agarose gel for use tomorrow. The size of the band I expect to find for the Tet FML is 349 bp (including the GCAT, BioBrick prefix, ATG, 5mer, and reporter gene until about 20 nucleotides or so past the restriction site we will cut at) and 474 bp for the RFP FML. According to the "Optimize Your Gel" link on the Davidson Lab Protocols page, a 1.6% gel will optimize how well I see my Tet bands and a 1.3% gel will optimize how well I see my RFP bands. I can use a gel with a concentration in between both of the optimal concentrations for Tet and RFP so that I can use one gel for both. Therefore, I will use a 1.45% gel. 1.45% of 60 uL buffer solution tells me that I should measure out 0.87 g agar for this gel.

== [[User:Roclemente|Roclemente]] 14:00, 18 June 2009 (EDT) ==

I checked on my PCR products this morning. Tubes 1 and 2 (both containing the RFP FML) had solution scattered on the tube's cover and walls so I placed them in the centrifuge for a bit. Unfortunately, I forgot to put these 500 uL tubes in holders when I centrifuged them so these tubes were destroyed. This was okay though because I was just going to combine each respective FML into one tube. I would have 300 uL of RFP FML PCR product instead of 500 uL and I put all of this in tube 5. I still had 500 uL of Tet FML PCR product left and put all of this in tube 10.

I ran 5 uL of both RFP and Tet FMLs on a gel, as well as a 1 kb molecular weight marker. The MW marker is in lane 1, the RFP is in lane 3, and the Tet is in lane 4 (Leland ran his PCR product in lane 2):

[[Image:2009-06-18 09hr 56min.jpg]]

I was expecting the RFP PCR product to be 474 bp long and the Tet PCR product to be 349 bp long. Both appear to come out a bit shorter, but Dr. Campbell and I agreed that we wouldn't count these sequences out because the bands were quite close to what we expected.

I proceeded to clean and concentrate these two sequences using the procedure outlined in the "Clean and Concentrate DNA (after PCR, before digestion)." Now I had 295 uL of the Tet sequence and 495 uL of the RFP sequence. Since the procedure says I can only have 800 uL in the column at a time, with DNA:Buffer at a ratio of 1:2, I mixed the two in a column in a series of two rounds using the following volumes:

[[Image:Clean.png]]

I Nanodropped my DNA after cleaning it and attained the following results:

[[Image:Nanome.png]]

I wanted the OD260 (Optical Density at 260) to be under 1.5, which it is for both. I lost quite a bit of Tet DNA in the process, and will only continue to lose DNA. To account for this loss of DNA, Dr. Campbell suggested it would be a good idea to heat inactivate the restriction enzymes to get rid of them after digestion instead of cleaning and concentrating the DNA over again.

I double digested both RFP and Tet FML sequences. I used all 9.5 uL of my DNA. The following table outlines how much volume of each material I mixed together:

[[Image:2nddoubledigest.png]]

While I waited 3 hours for my digestion to take place, I prepared 6 tubes to make more liquid cultures of the RFP and Tet vectors to grow more receiving vectors. 3 tubes would be to make RFP cultures and the other 3 would be for the Tet cultures. For RFP, I used the part I715039 and for Tet, I used the part J31007.

Once the digestion took place, Dr. Campbell notified me that heat inactivation was capable for Nco1 but not BamH1. There were 3 options for me to take in order to clean the Tet FML, the protocols for all of which can be found on the Davidson Lab Protocols link:

# [http://www.bio.davidson.edu/courses/Molbio/Protocols/Clean_Concentrate.html Clean and Concentrate DNA (after PCR, before digestion)]
# [http://www.bio.davidson.edu/courses/Molbio/Protocols/clean_short.html Ethanol Precipitate DNA (short protocol)]
# [http://www.bio.davidson.edu/courses/Molbio/Protocols/Qiagen_gelpure.html Qiagen QIAquick Gel Purification]

Although I could've chosen to do the heat inactivation for the RFP FML, I chose to keep the purifying technique in synch for consistency's sake. I chose to do the gel purification. While this process takes longer than the other two processes, I have found that gel purification generates the greatest yield and therefore chose it.

IGEM 2009 notebook

2009-06-18T20:39:02Z

ShPunjabi: /* Roclemente 14:00, 18 June 2009 (EDT) */

== [[User:Roclemente|Roclemente]] 16:54, 3 June 2009 (EDT) ==

Shamita and I are trying to find suitable reporter proteins to use. Yesterday, Leland and Alyndria were working on ways to insert the gene sequences into the plasmid. Upon seeing how they wanted to manipulated the reporter gene to include the logical clauses, we came up with a few criteria for the reporter genes we would use. The following criteria for genes are listed in order of the broadest aspect to look at to the narrowest aspect:

a) Doesn't contain restriction sites for the 4 restriction enzymes (EcoR1, Xbal, Spel, Pst1) used to cleave the Biobrick part out of the plasmid.

b) Contains 6 cutter restriction sites.

c) These restriction enzymes aren't blunt (cleave straight down at one spot).

d) These restriction sites are close to thge 5' (beginning) end of the sequence.

e) These enzymes are easiest to work with and cheapest.

We are finding the part numbers of the reporter genes we want to use (antibiotic resistance, fluorescence, ''LacZ'') through our own GCAT because we know these ones work. We are then locating these parts on the parts registry [http://www.partsregistry.org] website. We copied and pasted the gene sequences we obtained from the registry onto the ApE software [http://www.biology.utah.edu/jorgensen/wayned/ape/]. From here, we were able to generate a genetic map of each gene that outlined each restriction site that fit our criteria. We put each genetic map, alongside the part number used, into a Word document:

http://gcat.davidson.edu/GcatWiki/images/0/0e/Restriction_Site_Mapping_on_Reporter_Genes.doc

So it looks like we've changed directions. The team is looking towards the wet lab portion of our research more now. Instead of simply speculating about which reporter proteins we think will work best and which tRNA suppressors to use, we're going to physically test it out ourselves. A few tasks at hand in the second part of this afternoon:

1. What are the DNA gene sequences of the 12 different tRNA suppressors we want to use?

2. What is the sequence of the DNA that is cleaved off of both ends of the tRNA when it is transcribed?

We e-mailed Anderson to find out the exact tRNA gene sequences because we had a hard time finding this information everywhere else. Most papers we found describing the 5-base codon tRNA suppressors simply referred to Anderson's paper [http://gcat.davidson.edu/GcatWiki/images/6/6f/AndersonSchultz2002ChemBiol.pdf]. Anderson replied near the end of the day with the gene sequence. According to his email, the only difference between different tRNA genes is the anticodon loop. Therefore, if we just substitute all the anticodon loops in the invariable part of the sequence, we have our different tRNA sequences. We plan on using the Lancilator to help us break these tRNA sequences into smaller oligonucleotides that can anneal at around the same temperature because it is not possible for us to order oligos that are greater than 70 nucleotides long.

== [[User:Roclemente|Roclemente]] 17:21, 4 June 2009 (EDT) ==

I started out the day looking at my time-sensitive group research qustion about reporter proteins. I started making a table listing the advantages and disadvantages of different reporter proteins. Then the group found out that there was a major roadblock to our project: a stop codon was located in our ATG-5mer oligo. The stop codon would have been part of the BioBrick scar. The group looked at several ways to work around this problem. The idea that I looked most into was replacing the restriction sites that were used in the scar through standard assembly. According to the judging criteria for iGEM, I found that a team could possibly alter the standard assembly method as long as they wrote a letter to the iGEM judges explaining our plan in a detailed manner. From there, I set off to understand the BioBrick scar more by making a document highlighting the exact positions of the rsetriction sites on the BioBrick parts and how they were used to put more than one part together. I then found several other pairs of restriction sites that complemented one another and could be used in place of the standard scar between Xba1 and Spe1. At the end of the day, the group got the chance to speak with Dr. Campbell and he suggested a hybrid idea which would combine Davidson's restriction site idea with Missouri's PCR idea. I'm not quite sure of what this idea is exactly yet, but we'll all clarify our vague idea of it tomorrow morning after a talk with Dr. Eckdahl.

== [[User:Roclemente|Roclemente]] 17:13, 5 June 2009 (EDT) ==

This morning we began exploring different options for bypassing the stop codon problem from yesterday. If we wanted to make sure we were using the BioBrick parts in our GCatalog, there was no way we were going to be getting rid of the Xba1 restriction site that contained the "TAG" which codes for a stop codon. Therefore, we proposed looking at restriction sites within the reporter genes instead, therefore addressing the already-placed BioBrick ends. Using the document me and Shamita created on "Restriction Site Mapping on Reporter Genes" (see first journal entry), we determined that the reporter gene with the restriction site closest to the beginning of the sequence was YFP. We proposed cutting the YFP gene that we already had in stock at this restriction site and inserting an oligo (which we would have ordered) with a sequence in the following order: "BioBrick prefix - ATG - 5mer - part of YFP gene to the left of the cleaved restriction site."

After clarifying our own idea with one another, we spoke with some Missouri kids to clarify the said "hybrid" idea which, according to Dr. Campbell, combined the ideas of both campuses. The hybrid idea was based upon our idea of cutting the reporter gene at a restriction site as well, except that our primer would start out with a primer for our reporter gene, and to the left of the primer would be a "tail" consisting of the ATG and 5mer and either an Xba1 or EcoR1 restriction site (We still have to decide this. Our decision will probably be based on the restriction site we end up using in our reporter gene and whether or not that is complementary to either the Xba1 or EcoR1 sites.). We would use the PCR technique and use our in-stock reporter gene strand as our template strand (it will be denatured into single strands) and we will add our oligo. We propose that the primer will synthesize the rest of the YFP strand as well as add on nucleotides to complete the oligo tail. In the end, we will have a double stranded DNA sequence consisting of either the Xba1 or EcoR1 site, the ATG, the 5mer, and the reporter gene. The beauty of this hybrid idea is that we have flexibility as far as the reporter gene we choose to use since all we really need for it is the beginning sequence (for the primer) and a known, "hearty" restriction site. Once we cut this double strand at this restriction site on the reporter gene as well as at the Xba1 or EcoR1 sites, we can insert this into a plasmid that contains the rest of that reporter gene after the restriction site we choose to use and the complementary Xba1 or EcoR1 site.

I made a Word document outlining the promoter and RBS parts we have in our registry on the Davidson campus. Leland and I went through the registry parts on the GCATalog to look for potential promoters and RBS parts. We went through the document as a group and analyzed each of these potential parts on partsregistry.org to see which ones would most likely work the best in the miniprep we'll start preparing for on Sunday evening. Here is the document we wound up with:

http://gcat.davidson.edu/GcatWiki/images/d/d2/Promoters_and_RBS.doc

== [[User:Roclemente|Roclemente]] 10:40, 8 June 2009 (EDT) ==

We're starting the day by finalizing the oligos we will be ordering to have synthesized. Two groups of oligos must be sequenced: primers and tRNA gene sequence. The primers will consist of a forward and reverse primer. The forward primer will consist of the following in this 5' - 3' order: GCAT - BioBrick Prefix - ATF - 5mer - first 20 nucleotides in the reporter gene sequence. These 20 nucleotides won't include the ATG that naturally comes at the beginning of the reporter gene because we want to make sure the ribosome attaches to the first ATG and reads the logical clause instead of going straight to the ATG at the start of the reporter gene. The reverse primer will consist of the first 20 nucleotides downstream of the restriction site we plan to use in the reporter gene. I worked on finding the primers for the Tet resistance gene (part J31007) while Lyn worked on finding the primers for the RGP gene. Dr. Campbell decided we should use the BamH1 restriction enzyme because it is very "hardy." The following document outlines the 5 forward primers (each with one of the five variable 5mers) we will order for amplification of the Tet resistance FML:

[[Image:Tet resistance forward primers.png]]

This is the reverse primer we will order for the Tet resistance FML:

[[Image:Tet resistsance reverse primer.png]]

== [[User:Roclemente|Roclemente]] 11:54, 9 June 2009 (EDT) ==

We need to digest the plasmids at the EcoR1 and Pst siets. Everytime we're going to set up a digestion, we'll want the following in these volumes: 10x Buffer (2 uL), DNA (~ 3 uL), enzymes 1 and 2 (1 uL each), water (13 uL), and final volume of all of these put together should be 20 uL.

Some notes about digestion: Enzymes should make up 10% of the final volume. As a general rule of thumb, make sure the tip of your pipet is close to the surface of the solution. This is especially pertinent when gathering 1 microL of each of the enzymes. The most sensitive components are the enzymes, so they should go last.

We'll have a bunch of tubes with each 3 microL of the specific DNA. We'll make a cocktail in a bigger tube consisting of the buffer, 2 enzymes, and water. The rationale behind thissis that you'll be less likely to make a bigger mistake with a bigger volume (versus getting 1 microL of enzyme per specific DNA). You'll also know that there is no problem with the enzyme if all the DNA don't cut. If only one specific DNA doesn't work, then we'll know that it's because there's something wrong with the specific DNA rather than anything in the "Master Mix." Since we have to do 11 digestions, we'll make enough of the master mix for 12 tubes to make sure we have enough. We'll take 17 microL of the Master Mix to each tube of DNA.

The optimal temperature for most enzymes is 37 degreees celsius. You'd like to have enzymes with the same optimal temperatures (for ex. restriction enzymes that start with B usually have an optimal temperature near 50 degrees. You wouldn't want to use both B-enzyme and EcoR1 because EcoR1 would die).

'''What percent gel should we use?'''

Look at the Davidson lab protocol on the Davidson-Missouri Wiki. Generally, bigger molecules require an agarose gel with a lower concentration.

'''What's next in the future?'''

5' end additions are done by PCR. We'll ampliffy this double-stranded DNA. We'll cut with both EcoR1 and BamH1 or Nco1 (depending on which reporter gene you're using). Then we'll cut the plasmid with the reporter with the same restriction enzymes used.Then, to throw away the remaining DNA that we don't want, we'll run a gel and throw away the smaller band (because the larger band is probably made of the plasmid we want to keep).Then we'll take the double stranded DNA we amplified and the purified plasmid and ligate, transform, and screen them and verify the sequence.

While we wait for the oligos, we can cut the plasmid with reporter on the same restriction enzyme already.

'''How are we building the tRNAs?'''

We'll assemble the oligos into genes by mixing our oligos together, boiling them, and very slowly allowing them to cool. This will require many calculations. We'll cut the plasmids with EcoR1 and Pst1. to be ready to receive the enes we're assembling.We'll have to gel purify the plasmids to get them away from their inserts. We'll ligate, transform, and screen the genes and the plasmids and verify the sequence. On paper you shouldn't have two sites cut with different restriction enzymes stick together, but if there's selection pressure, these sites may cut some ends to fit together. This is why we must screen them.

After Dr. Campbell taught us all of the above, the 5 biologists put together a document outlining exactly how we would go about cutting the two sets of plasmids (one set will be cut with Eco R1 and Pst1 and othe other set will be cut with Eco R1 and BamH1 or Nco1). The following table outlines which biologist was going to work with which sample of DNA:

[[Image:Names and DNA.jpg]]

The following summarize the diferent materials and volumes of these materials that we'll need for our digestion of the plasmids:

[[Image:Digestion materials.png]]

This last table highlights the agarose percent concentration we should use for each part according to how many base pairs it contains:

[[Image:Screen-capture-3.png]]

The biologists digested the plasmids that we plan on using once our oligos arrive in a day or two. Afterwards, we made the two agarose gels that were most compatible with the parts we were going to run. This gel is going to purify the plasmids so that we have the parts that we're going to use later on, minus the rest of the DNA in the plasmid that we don't need. Before physically creating the gel, we figured out how much agarose we would need to make our two different agarose gel concentrations, 0.8% and 2.5%. Next, we made sure the mold and the big teeth were in place. We used the big teeth at the end of the mold because we plan on running 20 uL of DNA versus a running a smaller portion which would require us to use small teeth. We then measured the amount of agarose that we calculated we would need, placed this in a volumetric flask greater than 60 uL to allow for fizz to rise up, then measured out 60 uL of buffer and mixed this in the same flask with the agarose. We placed clear plastic wrap on top of the flask and heated it up for 1 minute and 20 seconds in the microwave. After we heated it, we took it out and checked to see if there was still agarose present that hadn't yet melted. There was, so we placed it back in the microwave and heated it for another 20 seconds. All the agarose had melted. We added 1 uL of EtBr (ethidium bromide) to the solution and quickly poured this into the mold. It needs about 15 to 20 minutes to cool. Dr. Campbell made the agarose gel that was 2.5% concentrated gel while we watched. We then made the 0.8% concentrated gel while he watched.

The following table lists the order in which we loaded our DNA into the gel. The first column all the way to the left is represented by number 1:

[[Image:Screen-capture-2.png]]

== [[User:Roclemente|Roclemente]] 08:46, 10 June 2009 (EDT) ==

After about 30 minutes after we began the gel electrophoresis, we looked at our gels with UV light to see how far the inserts had travelled and where the plasmids were. Then we took each gel to be photographed in the freezer room (231). We used the following DNA length marker to measure how long each sequence represented by each band was:

[[Image:Screen-capture-1.png]]

We compared our data to the lengths we expected to get according to the registry online. The first gel we photographed was the one that was 2.5% concentrated:

[[Image:2009-06-09 14hr 44min.jpg]]

The first two columns of the DNA we ran, both consisting of B0300, were fairly the same as the length we expected. However, the last two columns, consisting of K091111 and K091112 respectively were supposed to be 86 bp long, however, according to our data, K091112 was longer. Leland and I looked at Pallavi Penumetcha's paper which delved more closely into these two parts and what she wrote in her paper and what she entered into the registry conflict. We are currently corresponding with her to figure out the exact discrepancy.

We then photographed the 0.8% gel:

[[Image:2009-06-09 15hr 21min.jpg]]

The length of the sequences in almost every column matched up with the lengths we expected. Only the 4th and 5th columns didn't come out clearly. We think that the DNA inserted in column 5 weren't inserted properly and diffused on its own. We couldn't see bands in the 4th column at all until we adjusted the color a bit on the photographer and saw that column 4 was pretty much identical to column 5. We assumed that column 5 bled into column 4.

== [[User:Roclemente|Roclemente]] 15:14, 11 June 2009 (EDT) ==

We are digesting two parts today: I715039 (pLac+RBS+RFP) and S03710 (Tet Resistance). We want both the plasmids and the inserts in each case. We are aiming for a final volume of 40 uL for each part. This means that each digestion will consist of 4 uL buffer, 2 uL EcoR1, and 2 uL of their other respective restriction site (BamH1 for S03710 and Nco1 for I715039). The only parts that will vary are the amount of DNA and water we use. The amount of DNA we use depends on the concentration of the DNA we have (found through use of the NanoDrop). Since we want 2500 ng of DNA, we can use the concentration of DNA to figure out how much volume of this concentrated DNA we'll want to use in your digestion. If we subtract the volumes of buffer, two restriction enzymes, and DNA we plan on using from 40 uL, we'll have the amount of water we need to add. Here are the different volumes of materials we plan on using in our double digestion:

[[Image:DoubledigestRFPTetR.png]]

Now that we know how much of each material we were going to use, we needed to find out which buffer would be compatible with each sample according to the restriction enzymes we were using and how effective they'd be in different salt solutions. Online on the Promega "Compatible Buffers" website, we were able to find which Promega buffers would be most compatible when doing a double digest with EcoR1 and BamH1:

[[Image:BamEcoR1.png]]

Here is a chart noting buffers and their compatiblity with EcoR1 and Nco1:

[[Image:NcoEcoR1.png]]

We still have to wait for the restriction enzymes to arrive, so afterwards, we transformed three different inserts containing needed promoters and RBSs into vectors. We used the Zippy Transformation method. These promoters and RBSs will later be added to the FMLs once they are already inserted into the reporter cells. The following table outlines these inserts and part numbers:

[[Image:Promoterinsertspartnumbers.png]]

The restriction enzymes have arrived. We will now be able to do PCR to amplify the RFP gene (I715039) and the Tet resistance gene (S03710) as templates. We will be using the primers which have also arrived. We ordered forward and reverse primers so that our amplified sequence will consist of the following in this order:

GCAT-ATG-5mer-Reporter Gene

The following table lists the materials will be needed for this PCR technique which uses the "Green Promega master (Monster) Mix:"

[[Image:Greenpromegamastermix.png]]

This is the PCR procedure:

[[Image:Temperaturecycle.png]]

These are the variables that we will be adding to each separate sequence we want to be amplified:

[[Image:Tobeamplified.png]]

This table shows how we labeled the tubes based on how we divided the PCR work for the 10 tubes:

[[Image:Pcrtubes.png]]

Now we are giong to assemble the tRNA oligos together to form the tRNA genes. We could only order a certain length of sequence and the tRNA genes outdid these specified lengths. We used the Lancilator to find various oligonucleotides that overlapped and would be able to anneal together to form the tRNA genes. We used the Davidson lab protocol entitled "Buiding dsDNA with Oligos" to assemble the oligos:

[[Image:Oligoassembly.png]]

== [[User:Roclemente|Roclemente]] 22:46, 12 June 2009 (EDT) ==

We had two main goals for today:
1. perform a tRNA ligation and transformation
2. perform two electrophoreses: one to verify the PCR sequences we obtained and another to purify the digested reporter plasmids we would need

We began the day by pouring the agarose gels according to which concentration of buffer would be most compatible with the molecular weight of the substance we wanted to isolate. For both the digestion of E01010 and J31007, according to the "Optimize your Gel" website found on the Davidson Lab Protocols site for iGEM 2009, we wanted a 0.4% concentrated gel. For the PCRs, we wanted a 1.6% concentrated gel to optimize our retrieval of the Tet gene and a 1.4% concentrated gel to optimize our retrieval of the RFP gene.

While we waited for the gels to harden for about 30 minutes, we began calculating how much of each material we would need to ligate our tRNAs. We used the following equation to find out how much insert we should use:

[[Image:Ligationequation.png]]

Using the lengths of sequences we would be ligating, this is the mass that we needed:

X nanograms of insert = [(2)(144bp)(50 ng linearalized plasmid)]/2038bp
X = 7.0657 ng of insert

The following table outlines our calculations for finding the mass of tRNAs we had:

[[Image:Ligationcalculations.png]]

We would attain this mass of tRNA by pipetting an unknown amount of it. In order to attain a more "pipetable" amount of tRNA, we diluted our initial concentration 50X. Since we knew we wanted our final mass to be 7.065 ng, we calculated the volume we would need to take from this new, diluted sample:

DILUTION: 555.48/50 = 11.012
CALCULATION: 11.012 ng/ 1uL = 7.065 ng/ Y uL
Y = .64 uL of insert

Afterwards, Olivia and Alyndria began the actual ligation process while Shamita, Leland, and I filled the wells of the two gels. The first included the two digested reporter parts. We also included a molecular weight marker. Here is a photograph of this gel:

[[Image:2009-06-12 15hr 54min.jpg]]

According to our prior calculations, the Tet plasmid should have been 2958 base pairs long and the RFP plasmid should have been 2380 base pairs long. Our expectations were verified by the data. Shamita and I then sliced the part of thsi gel containing both plasmids and put these slices away to purfiy on Monday.

The second gel contained aliquots of the PCR we had done to attain 10 FML sequences: 5 containing the Tet resistant reporter gene and each of the 5 frameshifts that Davidson was assigned and the other 5 containing the RFP reporter gene and each of the 5 frameshifts as well:

[[Image:2009-06-12 16hr 07min.jpg]]

According to our data, each sequence containing the Tet resistant gene should have been 317 base pairs long and each sequence containing the RFP gene should have been 446 base pairs long. Our data supports our hypothesis, however, the 3rd well was not successful. This well contained the FML consisting of the Tet resistant gene and the frameshift mutant CUAGU. Before leaving work for the day, Shamita and I prepared and performed PCR using two parts containing the tet resistant gene, J31007 and S03710. We wanted to use both to make sure at least one of them works since we could technically use either of them, even though when we first did PCR we only used the S03710 part. Using the known concentrations of each of these parts, and knowing that we needed 1 ng of each part to include in our PCR samples, we calculated the volume of each 100X-diluted solution we would need:

S03710: 1.636 ng/uL = 1 ng/ X uL
X = 0.611 uL

J30017: 0.776 ng/uL = 1 ng/ Y uL
Y = 1.289 uL

We will run another gel with both of these parts on Monday and hopefully at least one of them works.

== [[User:Roclemente|Roclemente]] 17:24, 15 June 2009 (EDT) ==

Each biology member of the Davidson iGEM team has been assigned a 5mer to carry on throughout the rest of their respective processes. I've been assigned the 5mer CUAGU. We verified the PCR of the CUAGU FML containing the RFP gene on Friday. However, we weren't able to verify the CUAGU FML containing the tet resistance gene when we ran it on a gel. Shamita and I redid PCR for two parts containing the tet resistance gene: J31007 and S03710, labeled 2* and 2! respectively. I'm running another gel to verify either or both of them. Both sequences should be 317 bp long (this includes the ATF, logical clause, and the tet resistance gene to the left of the BamH1 restriction site). Here is the gel run we collected. The first column represents the 1 kb DNA marker, and the next two are 2* then 2!:

[[Image:2009-06-15 10hr 34min.jpg]]

The data shows that the PCRs of both parts wound up at the length we expected. The first time we did PCR on J31007, we must have left a solution out of our procedure on accident. I combined both PCRs together since they are the same length and match the length we expected to get.

Next, we cleaned and concentrated our amplified DNA sequence (#D4014 from Zymo Research). I cleaned the two pieces of DNA that contained the 5mer CUAGU. I labeled the tube with DNA also containing the RFP gene as "1" and the tube with DNA also containing the tet resistance gene as "2!". We needed 2 volumes of buffer for every 1 volume of DNA we added. Therefore, for tube "1," I added 95 uL DNA and 190 uL buffer. I added more DNA to tube "2!" since I combined the DNA from two PCR events to one (2* and 2! from earlier).

After cleaning the DNA, we found its concentration using the Nanodrop. In a solution made up of 0.5 uL of the DNA and 2.5 uL of TE, we found the following data from the DNA in this solution:

[[Image:Cleanconcentrate.png]]

The concentration of the DNA we cleaned was the concentration given by the Nanodrop multiplied by 6 (the DNA was dilute 6-fold for the Nanodrop). We used this concentration and the volume of clean DNA we knew we had (9.5 uL) to find the mass of clean DNA we had:

[[Image:Concentrateddna.png]]

Before doing digestion, we used the ligation equation (see "Ligation Protocol" in "Davidson Lab Protocols") to figure out how much mass of the DNA insert we would need later on when we did ligation. For the RFP FML, we found we would want the following mass of insert:

ng of insert = (2)(443)(50)/(2320) = 19.0948 ng

We multiplied this mass by 50 because we're counting on losing DNA between the digestion and cleaning we would need to do leading up to the ligation protocol.

19.0948 ng x 50 = 954.741 ng RFP FML

We found the mass of Tet FML insert we would need for ligation as well:

ng of insert = (2)(314)(50)/(2958) = 10.6153 ng

We multiplied this mass by 50 too:

10.6153 x 50 = 530.764 ng Tet FML

Unfortunately, nobody had enough of their clean DNA to satisfy our careful criteria. However, we would need to do this calculation later on so it was helpful we did it anyway. Instead, we just used all the DNA we had, or 9.5 uL for each reporter gene. Since there were 4 people who needed the same master mix, we made 5 parts of the master mix using the following volumes:

[[Image:Rfptetdigestion.png]]

After realizing that we would have to wait a day for the digestion to take place, we decided to culture every tRNA cell that colonized. The tRNA that we transformed on Friday did not have a good yield, so last night a few people came in and transformed more tRNA cells. We got a few more colonies. To be safe and make sure we cultured each tRNA, we picked every colony visible to us in both the old and new plates. Using our own given tRNAs, I counted the amount of colonies that grew in the plate we transformed on 6/12 and 6/14. There were 4 colonies that grew on both plates so I labeled a tube for each of these. I awas also in charge of the negative control. There were 2 colonies that grew on the negative control plate so I labeled 2 tubes for that. We will be placing them in an incubator overnight.

== [[User:Roclemente|Roclemente]] 12:30, 16 June 2009 (EDT) ==

We checked on the cells we cultured overnight. Of the cells that I cultured (all containing the CUAGU 5mer and the two negative controls), both negative control tubes worked as well as the tubes I labeled 1 (6/14), 5 (6/12), and 7 (6/12). The rest of mine were either red, meaning the plasmids hadn't ligated properly) or the colonies we used were no good. I took these 5 tubes, along with 3 of Olivia's tubes filled with cells that were cultured (tubes 11, 12, and 13) and miniprepped them to isolate the plasmids from the cells. There was no need to colony PCR to screen for successful ligations because this is only used to know which ones to let grow overnight and we had already done this.

As soon as I finished miniprepping them, I had to leave the lab for a little bit, so Olivia took each my miniprep tubes besides the negative controls and Leland took my negative controls. I completed the protocol for digestion of the tRNAs with EcoR1 and Pst1 for Alyndria's tubes when I got back because she had left a little earlier for lunch.

Afterwards, Shamita and I poured a 1.9% concentrated gel to load the digested tRNAs on. We want to verify that these tRNAs are the appropriate size for if they were cut properly. Since we will have to load 37 wells, and the maximum number we can load on one gel is 18 since 2 wells will have to be used by the molecular weight marker, we needed to pour 2 gels and we will also be using a half of a 2.0% agarose gel that was left over from another experiment.

Shamita and I also made new 0.5X running buffer gel. We did this by adding 450 uL buffer and 9 uL ethidium bromide (1 uL EtBr : 50 uL buffer). Alyndria, Leland, and Shamita loaded these gels.

Olivia and I cleaned our PCR DNA using the "Ethanol precipitate DNA (short protocol)" found in the Davidson Lab Protocols link. To begin with, I added 180 uL dH2O to my two samples, CUAGU with RFP and CUAGU with Tet, to bring their volumes up to 200 uL. During one of the last steps of this protocol, when Olivia and I dried out our DNA using the SpeedVac for a period of 10 minutes, the top of my tube containing the CUAGU 5mer and RFP gene broke. I wasn't sure if a pellet was in there, but I still poured out the ethanol and went on with the protocol. At the end, I resuspended what may or may not have been my DNA in 10 uL distilled water. I NanoDropped it afterward and attained the following data concerning both tubes:

[[Image:Failedpcrmeh.png]]

According to this data, any absorption and concentration we may have attained is insignificant because the A-260 must be between 0.05 and 1.50 to be significant and neither of the DNA is. Tomorrow I will do PCR for both FMLs over again.

== [[User:Roclemente|Roclemente]] 14:51, 17 June 2009 (EDT) ==

I found out that my tRNA DNA that I labeled "1" and which contains CUAGU was verified from the gel run Olivia did yesterday (the gel run also included her tRNA DNA samples). At the start of the day I plated this tube of cultured cells again to grow overnight.

Since PCR will take about 3 1/2 hours, I attempted this procedure at the beginning of the day. I plan on making 5 PCR products for each reporter gene since I now know the environmental conditions are correct. I labeled the RFP tubes 1-5 and the Tet tubes 6-10. I first needed to figure out how much of the template DNA I would need in order to have 1 ng template DNA. I made the following calculations to find this out:

[[Image:1ngtemplatedna.png]]

I wanted to add the Monster Mix last, so I made the following table, adding everything else besides the Monster Mix for 5 of the same reporter gene to one tube:

[[Image:Pcrmix.png]]

Unfortunately, when I actually mixed the solution together, I forgot to dilute the template DNA. Therefore, we have 100X more template DNA than we would have had we diluted the template DNA. The PCR can still work, but we will have to constantly remind ourselves that there will be extra template DNA in our solution and that this may skew, however slightly, our results later on.

While I waited for the PCR product, I refocused on getting my tRNA DNA ready for sequencing. Using the NanoDrop, I found the concentration of my miniprepped CUAGU DNA that was verified to be 61 ng/uL. The pre-sequencing protocol calls for 60 ng DNA, so I extracted 1 uL from this tube (the extra DNA will be insignificant in the sequencing procedure). I pipetted this into another tube and used the SpeedVac to dry it out. This is how we will send our DNA to be sequenced.

The following is a summary of steps I put together to help me plan out what I will do after the PCR procedure is over (it will be done at 5 pm today):

1. Combine the five matching tubes into one tube.
2. Run each PCR product on a gel to verify that our expected sequence has been properly amplified.
3. Clean and concentrate plasmids so salt solution is optimal for digestion.
4. Double digest PCR products with EcoR1 and BamH1 or Nco1.
5. Clean and concentrate again to get rid of restriction enzymes.
6. Nanodrop inserts in order to do ligation calculations and verify that sequences are present.
7. Ligate FML inserts with purified plasmids.
8. Transform plasmid into cells.

Since I won't have time to run a gel and take a picture once the PCR procedure is complete (we get out at 5:30 and it will be finished at 5:00), I have begun to prepare the agarose gel for use tomorrow. The size of the band I expect to find for the Tet FML is 349 bp (including the GCAT, BioBrick prefix, ATG, 5mer, and reporter gene until about 20 nucleotides or so past the restriction site we will cut at) and 474 bp for the RFP FML. According to the "Optimize Your Gel" link on the Davidson Lab Protocols page, a 1.6% gel will optimize how well I see my Tet bands and a 1.3% gel will optimize how well I see my RFP bands. I can use a gel with a concentration in between both of the optimal concentrations for Tet and RFP so that I can use one gel for both. Therefore, I will use a 1.45% gel. 1.45% of 60 uL buffer solution tells me that I should measure out 0.87 g agar for this gel.

== [[User:Roclemente|Roclemente]] 14:00, 18 June 2009 (EDT) ==

I checked on my PCR products this morning. Tubes 1 and 2 (both containing the RFP FML) had solution scattered on the tube's cover and walls so I placed them in the centrifuge for a bit. Unfortunately, I forgot to put these 500 uL tubes in holders when I centrifuged them so these tubes were destroyed. This was okay though because I was just going to combine each respective FML into one tube. I would have 300 uL of RFP FML PCR product instead of 500 uL and I put all of this in tube 5. I still had 500 uL of Tet FML PCR product left and put all of this in tube 10.

I ran 5 uL of both RFP and Tet FMLs on a gel, as well as a 1 kb molecular weight marker. The MW marker is in lane 1, the RFP is in lane 3, and the Tet is in lane 4 (Leland ran his PCR product in lane 2):

[[Image:2009-06-18 09hr 56min.jpg]]

I was expecting the RFP PCR product to be 474 bp long and the Tet PCR product to be 349 bp long. Both appear to come out a bit shorter, but Dr. Campbell and I agreed that we wouldn't count these sequences out because the bands were quite close to what we expected.

I proceeded to clean and concentrate these two sequences using the procedure outlined in the "Clean and Concentrate DNA (after PCR, before digestion)." Now I had 295 uL of the Tet sequence and 495 uL of the RFP sequence. Since the procedure says I can only have 800 uL in the column at a time, with DNA:Buffer at a ratio of 1:2, I mixed the two in a column in a series of two rounds using the following volumes:

[[Image:Clean.png]]

I Nanodropped my DNA after cleaning it and attained the following results:

[[Image:Nanome.png]]

I wanted the OD260 (Optical Density at 260) to be under 1.5, which it is for both. I lost quite a bit of Tet DNA in the process, and will only continue to lose DNA. To account for this loss of DNA, Dr. Campbell suggested it would be a good idea to heat inactivate the restriction enzymes to get rid of them after digestion instead of cleaning and concentrating the DNA over again.

I double digested both RFP and Tet FML sequences. I used all 9.5 uL of my DNA. The following table outlines how much volume of each material I mixed together:

[[Image:2nddoubledigest.png]]

While I waited 3 hours for my digestion to take place, I prepared 6 tubes to make more liquid cultures of the RFP and Tet vectors to grow more receiving vectors. 3 tubes would be to make RFP cultures and the other 3 would be for the Tet cultures. For RFP, I used the part I715039 and for Tet, I used the part J31007.

Once the digestion took place, Dr. Campbell notified me that heat inactivation was capable for Nco1 but not BamH1. There were 3 options for me to take in order to clean the Tet FML, the protocols for all of which can be found on the Davidson Lab Protocols link:

# [http://www.bio.davidson.edu/courses/Molbio/Protocols/Clean_Concentrate.html Clean and Concentrate DNA (after PCR, before digestion)]
# [http://www.bio.davidson.edu/courses/Molbio/Protocols/clean_short.html Ethanol Precipitate DNA (short protocol)]
# [http://www.bio.davidson.edu/courses/Molbio/Protocols/Qiagen_gelpure.html Qiagen QIAquick Gel Purification]

IGEM 2009 notebook

2009-06-18T20:38:36Z

ShPunjabi: /* Roclemente 14:00, 18 June 2009 (EDT) */

== [[User:Roclemente|Roclemente]] 16:54, 3 June 2009 (EDT) ==

Shamita and I are trying to find suitable reporter proteins to use. Yesterday, Leland and Alyndria were working on ways to insert the gene sequences into the plasmid. Upon seeing how they wanted to manipulated the reporter gene to include the logical clauses, we came up with a few criteria for the reporter genes we would use. The following criteria for genes are listed in order of the broadest aspect to look at to the narrowest aspect:

a) Doesn't contain restriction sites for the 4 restriction enzymes (EcoR1, Xbal, Spel, Pst1) used to cleave the Biobrick part out of the plasmid.

b) Contains 6 cutter restriction sites.

c) These restriction enzymes aren't blunt (cleave straight down at one spot).

d) These restriction sites are close to thge 5' (beginning) end of the sequence.

e) These enzymes are easiest to work with and cheapest.

We are finding the part numbers of the reporter genes we want to use (antibiotic resistance, fluorescence, ''LacZ'') through our own GCAT because we know these ones work. We are then locating these parts on the parts registry [http://www.partsregistry.org] website. We copied and pasted the gene sequences we obtained from the registry onto the ApE software [http://www.biology.utah.edu/jorgensen/wayned/ape/]. From here, we were able to generate a genetic map of each gene that outlined each restriction site that fit our criteria. We put each genetic map, alongside the part number used, into a Word document:

http://gcat.davidson.edu/GcatWiki/images/0/0e/Restriction_Site_Mapping_on_Reporter_Genes.doc

So it looks like we've changed directions. The team is looking towards the wet lab portion of our research more now. Instead of simply speculating about which reporter proteins we think will work best and which tRNA suppressors to use, we're going to physically test it out ourselves. A few tasks at hand in the second part of this afternoon:

1. What are the DNA gene sequences of the 12 different tRNA suppressors we want to use?

2. What is the sequence of the DNA that is cleaved off of both ends of the tRNA when it is transcribed?

We e-mailed Anderson to find out the exact tRNA gene sequences because we had a hard time finding this information everywhere else. Most papers we found describing the 5-base codon tRNA suppressors simply referred to Anderson's paper [http://gcat.davidson.edu/GcatWiki/images/6/6f/AndersonSchultz2002ChemBiol.pdf]. Anderson replied near the end of the day with the gene sequence. According to his email, the only difference between different tRNA genes is the anticodon loop. Therefore, if we just substitute all the anticodon loops in the invariable part of the sequence, we have our different tRNA sequences. We plan on using the Lancilator to help us break these tRNA sequences into smaller oligonucleotides that can anneal at around the same temperature because it is not possible for us to order oligos that are greater than 70 nucleotides long.

== [[User:Roclemente|Roclemente]] 17:21, 4 June 2009 (EDT) ==

I started out the day looking at my time-sensitive group research qustion about reporter proteins. I started making a table listing the advantages and disadvantages of different reporter proteins. Then the group found out that there was a major roadblock to our project: a stop codon was located in our ATG-5mer oligo. The stop codon would have been part of the BioBrick scar. The group looked at several ways to work around this problem. The idea that I looked most into was replacing the restriction sites that were used in the scar through standard assembly. According to the judging criteria for iGEM, I found that a team could possibly alter the standard assembly method as long as they wrote a letter to the iGEM judges explaining our plan in a detailed manner. From there, I set off to understand the BioBrick scar more by making a document highlighting the exact positions of the rsetriction sites on the BioBrick parts and how they were used to put more than one part together. I then found several other pairs of restriction sites that complemented one another and could be used in place of the standard scar between Xba1 and Spe1. At the end of the day, the group got the chance to speak with Dr. Campbell and he suggested a hybrid idea which would combine Davidson's restriction site idea with Missouri's PCR idea. I'm not quite sure of what this idea is exactly yet, but we'll all clarify our vague idea of it tomorrow morning after a talk with Dr. Eckdahl.

== [[User:Roclemente|Roclemente]] 17:13, 5 June 2009 (EDT) ==

This morning we began exploring different options for bypassing the stop codon problem from yesterday. If we wanted to make sure we were using the BioBrick parts in our GCatalog, there was no way we were going to be getting rid of the Xba1 restriction site that contained the "TAG" which codes for a stop codon. Therefore, we proposed looking at restriction sites within the reporter genes instead, therefore addressing the already-placed BioBrick ends. Using the document me and Shamita created on "Restriction Site Mapping on Reporter Genes" (see first journal entry), we determined that the reporter gene with the restriction site closest to the beginning of the sequence was YFP. We proposed cutting the YFP gene that we already had in stock at this restriction site and inserting an oligo (which we would have ordered) with a sequence in the following order: "BioBrick prefix - ATG - 5mer - part of YFP gene to the left of the cleaved restriction site."

After clarifying our own idea with one another, we spoke with some Missouri kids to clarify the said "hybrid" idea which, according to Dr. Campbell, combined the ideas of both campuses. The hybrid idea was based upon our idea of cutting the reporter gene at a restriction site as well, except that our primer would start out with a primer for our reporter gene, and to the left of the primer would be a "tail" consisting of the ATG and 5mer and either an Xba1 or EcoR1 restriction site (We still have to decide this. Our decision will probably be based on the restriction site we end up using in our reporter gene and whether or not that is complementary to either the Xba1 or EcoR1 sites.). We would use the PCR technique and use our in-stock reporter gene strand as our template strand (it will be denatured into single strands) and we will add our oligo. We propose that the primer will synthesize the rest of the YFP strand as well as add on nucleotides to complete the oligo tail. In the end, we will have a double stranded DNA sequence consisting of either the Xba1 or EcoR1 site, the ATG, the 5mer, and the reporter gene. The beauty of this hybrid idea is that we have flexibility as far as the reporter gene we choose to use since all we really need for it is the beginning sequence (for the primer) and a known, "hearty" restriction site. Once we cut this double strand at this restriction site on the reporter gene as well as at the Xba1 or EcoR1 sites, we can insert this into a plasmid that contains the rest of that reporter gene after the restriction site we choose to use and the complementary Xba1 or EcoR1 site.

I made a Word document outlining the promoter and RBS parts we have in our registry on the Davidson campus. Leland and I went through the registry parts on the GCATalog to look for potential promoters and RBS parts. We went through the document as a group and analyzed each of these potential parts on partsregistry.org to see which ones would most likely work the best in the miniprep we'll start preparing for on Sunday evening. Here is the document we wound up with:

http://gcat.davidson.edu/GcatWiki/images/d/d2/Promoters_and_RBS.doc

== [[User:Roclemente|Roclemente]] 10:40, 8 June 2009 (EDT) ==

We're starting the day by finalizing the oligos we will be ordering to have synthesized. Two groups of oligos must be sequenced: primers and tRNA gene sequence. The primers will consist of a forward and reverse primer. The forward primer will consist of the following in this 5' - 3' order: GCAT - BioBrick Prefix - ATF - 5mer - first 20 nucleotides in the reporter gene sequence. These 20 nucleotides won't include the ATG that naturally comes at the beginning of the reporter gene because we want to make sure the ribosome attaches to the first ATG and reads the logical clause instead of going straight to the ATG at the start of the reporter gene. The reverse primer will consist of the first 20 nucleotides downstream of the restriction site we plan to use in the reporter gene. I worked on finding the primers for the Tet resistance gene (part J31007) while Lyn worked on finding the primers for the RGP gene. Dr. Campbell decided we should use the BamH1 restriction enzyme because it is very "hardy." The following document outlines the 5 forward primers (each with one of the five variable 5mers) we will order for amplification of the Tet resistance FML:

[[Image:Tet resistance forward primers.png]]

This is the reverse primer we will order for the Tet resistance FML:

[[Image:Tet resistsance reverse primer.png]]

== [[User:Roclemente|Roclemente]] 11:54, 9 June 2009 (EDT) ==

We need to digest the plasmids at the EcoR1 and Pst siets. Everytime we're going to set up a digestion, we'll want the following in these volumes: 10x Buffer (2 uL), DNA (~ 3 uL), enzymes 1 and 2 (1 uL each), water (13 uL), and final volume of all of these put together should be 20 uL.

Some notes about digestion: Enzymes should make up 10% of the final volume. As a general rule of thumb, make sure the tip of your pipet is close to the surface of the solution. This is especially pertinent when gathering 1 microL of each of the enzymes. The most sensitive components are the enzymes, so they should go last.

We'll have a bunch of tubes with each 3 microL of the specific DNA. We'll make a cocktail in a bigger tube consisting of the buffer, 2 enzymes, and water. The rationale behind thissis that you'll be less likely to make a bigger mistake with a bigger volume (versus getting 1 microL of enzyme per specific DNA). You'll also know that there is no problem with the enzyme if all the DNA don't cut. If only one specific DNA doesn't work, then we'll know that it's because there's something wrong with the specific DNA rather than anything in the "Master Mix." Since we have to do 11 digestions, we'll make enough of the master mix for 12 tubes to make sure we have enough. We'll take 17 microL of the Master Mix to each tube of DNA.

The optimal temperature for most enzymes is 37 degreees celsius. You'd like to have enzymes with the same optimal temperatures (for ex. restriction enzymes that start with B usually have an optimal temperature near 50 degrees. You wouldn't want to use both B-enzyme and EcoR1 because EcoR1 would die).

'''What percent gel should we use?'''

Look at the Davidson lab protocol on the Davidson-Missouri Wiki. Generally, bigger molecules require an agarose gel with a lower concentration.

'''What's next in the future?'''

5' end additions are done by PCR. We'll ampliffy this double-stranded DNA. We'll cut with both EcoR1 and BamH1 or Nco1 (depending on which reporter gene you're using). Then we'll cut the plasmid with the reporter with the same restriction enzymes used.Then, to throw away the remaining DNA that we don't want, we'll run a gel and throw away the smaller band (because the larger band is probably made of the plasmid we want to keep).Then we'll take the double stranded DNA we amplified and the purified plasmid and ligate, transform, and screen them and verify the sequence.

While we wait for the oligos, we can cut the plasmid with reporter on the same restriction enzyme already.

'''How are we building the tRNAs?'''

We'll assemble the oligos into genes by mixing our oligos together, boiling them, and very slowly allowing them to cool. This will require many calculations. We'll cut the plasmids with EcoR1 and Pst1. to be ready to receive the enes we're assembling.We'll have to gel purify the plasmids to get them away from their inserts. We'll ligate, transform, and screen the genes and the plasmids and verify the sequence. On paper you shouldn't have two sites cut with different restriction enzymes stick together, but if there's selection pressure, these sites may cut some ends to fit together. This is why we must screen them.

After Dr. Campbell taught us all of the above, the 5 biologists put together a document outlining exactly how we would go about cutting the two sets of plasmids (one set will be cut with Eco R1 and Pst1 and othe other set will be cut with Eco R1 and BamH1 or Nco1). The following table outlines which biologist was going to work with which sample of DNA:

[[Image:Names and DNA.jpg]]

The following summarize the diferent materials and volumes of these materials that we'll need for our digestion of the plasmids:

[[Image:Digestion materials.png]]

This last table highlights the agarose percent concentration we should use for each part according to how many base pairs it contains:

[[Image:Screen-capture-3.png]]

The biologists digested the plasmids that we plan on using once our oligos arrive in a day or two. Afterwards, we made the two agarose gels that were most compatible with the parts we were going to run. This gel is going to purify the plasmids so that we have the parts that we're going to use later on, minus the rest of the DNA in the plasmid that we don't need. Before physically creating the gel, we figured out how much agarose we would need to make our two different agarose gel concentrations, 0.8% and 2.5%. Next, we made sure the mold and the big teeth were in place. We used the big teeth at the end of the mold because we plan on running 20 uL of DNA versus a running a smaller portion which would require us to use small teeth. We then measured the amount of agarose that we calculated we would need, placed this in a volumetric flask greater than 60 uL to allow for fizz to rise up, then measured out 60 uL of buffer and mixed this in the same flask with the agarose. We placed clear plastic wrap on top of the flask and heated it up for 1 minute and 20 seconds in the microwave. After we heated it, we took it out and checked to see if there was still agarose present that hadn't yet melted. There was, so we placed it back in the microwave and heated it for another 20 seconds. All the agarose had melted. We added 1 uL of EtBr (ethidium bromide) to the solution and quickly poured this into the mold. It needs about 15 to 20 minutes to cool. Dr. Campbell made the agarose gel that was 2.5% concentrated gel while we watched. We then made the 0.8% concentrated gel while he watched.

The following table lists the order in which we loaded our DNA into the gel. The first column all the way to the left is represented by number 1:

[[Image:Screen-capture-2.png]]

== [[User:Roclemente|Roclemente]] 08:46, 10 June 2009 (EDT) ==

After about 30 minutes after we began the gel electrophoresis, we looked at our gels with UV light to see how far the inserts had travelled and where the plasmids were. Then we took each gel to be photographed in the freezer room (231). We used the following DNA length marker to measure how long each sequence represented by each band was:

[[Image:Screen-capture-1.png]]

We compared our data to the lengths we expected to get according to the registry online. The first gel we photographed was the one that was 2.5% concentrated:

[[Image:2009-06-09 14hr 44min.jpg]]

The first two columns of the DNA we ran, both consisting of B0300, were fairly the same as the length we expected. However, the last two columns, consisting of K091111 and K091112 respectively were supposed to be 86 bp long, however, according to our data, K091112 was longer. Leland and I looked at Pallavi Penumetcha's paper which delved more closely into these two parts and what she wrote in her paper and what she entered into the registry conflict. We are currently corresponding with her to figure out the exact discrepancy.

We then photographed the 0.8% gel:

[[Image:2009-06-09 15hr 21min.jpg]]

The length of the sequences in almost every column matched up with the lengths we expected. Only the 4th and 5th columns didn't come out clearly. We think that the DNA inserted in column 5 weren't inserted properly and diffused on its own. We couldn't see bands in the 4th column at all until we adjusted the color a bit on the photographer and saw that column 4 was pretty much identical to column 5. We assumed that column 5 bled into column 4.

== [[User:Roclemente|Roclemente]] 15:14, 11 June 2009 (EDT) ==

We are digesting two parts today: I715039 (pLac+RBS+RFP) and S03710 (Tet Resistance). We want both the plasmids and the inserts in each case. We are aiming for a final volume of 40 uL for each part. This means that each digestion will consist of 4 uL buffer, 2 uL EcoR1, and 2 uL of their other respective restriction site (BamH1 for S03710 and Nco1 for I715039). The only parts that will vary are the amount of DNA and water we use. The amount of DNA we use depends on the concentration of the DNA we have (found through use of the NanoDrop). Since we want 2500 ng of DNA, we can use the concentration of DNA to figure out how much volume of this concentrated DNA we'll want to use in your digestion. If we subtract the volumes of buffer, two restriction enzymes, and DNA we plan on using from 40 uL, we'll have the amount of water we need to add. Here are the different volumes of materials we plan on using in our double digestion:

[[Image:DoubledigestRFPTetR.png]]

Now that we know how much of each material we were going to use, we needed to find out which buffer would be compatible with each sample according to the restriction enzymes we were using and how effective they'd be in different salt solutions. Online on the Promega "Compatible Buffers" website, we were able to find which Promega buffers would be most compatible when doing a double digest with EcoR1 and BamH1:

[[Image:BamEcoR1.png]]

Here is a chart noting buffers and their compatiblity with EcoR1 and Nco1:

[[Image:NcoEcoR1.png]]

We still have to wait for the restriction enzymes to arrive, so afterwards, we transformed three different inserts containing needed promoters and RBSs into vectors. We used the Zippy Transformation method. These promoters and RBSs will later be added to the FMLs once they are already inserted into the reporter cells. The following table outlines these inserts and part numbers:

[[Image:Promoterinsertspartnumbers.png]]

The restriction enzymes have arrived. We will now be able to do PCR to amplify the RFP gene (I715039) and the Tet resistance gene (S03710) as templates. We will be using the primers which have also arrived. We ordered forward and reverse primers so that our amplified sequence will consist of the following in this order:

GCAT-ATG-5mer-Reporter Gene

The following table lists the materials will be needed for this PCR technique which uses the "Green Promega master (Monster) Mix:"

[[Image:Greenpromegamastermix.png]]

This is the PCR procedure:

[[Image:Temperaturecycle.png]]

These are the variables that we will be adding to each separate sequence we want to be amplified:

[[Image:Tobeamplified.png]]

This table shows how we labeled the tubes based on how we divided the PCR work for the 10 tubes:

[[Image:Pcrtubes.png]]

Now we are giong to assemble the tRNA oligos together to form the tRNA genes. We could only order a certain length of sequence and the tRNA genes outdid these specified lengths. We used the Lancilator to find various oligonucleotides that overlapped and would be able to anneal together to form the tRNA genes. We used the Davidson lab protocol entitled "Buiding dsDNA with Oligos" to assemble the oligos:

[[Image:Oligoassembly.png]]

== [[User:Roclemente|Roclemente]] 22:46, 12 June 2009 (EDT) ==

We had two main goals for today:
1. perform a tRNA ligation and transformation
2. perform two electrophoreses: one to verify the PCR sequences we obtained and another to purify the digested reporter plasmids we would need

We began the day by pouring the agarose gels according to which concentration of buffer would be most compatible with the molecular weight of the substance we wanted to isolate. For both the digestion of E01010 and J31007, according to the "Optimize your Gel" website found on the Davidson Lab Protocols site for iGEM 2009, we wanted a 0.4% concentrated gel. For the PCRs, we wanted a 1.6% concentrated gel to optimize our retrieval of the Tet gene and a 1.4% concentrated gel to optimize our retrieval of the RFP gene.

While we waited for the gels to harden for about 30 minutes, we began calculating how much of each material we would need to ligate our tRNAs. We used the following equation to find out how much insert we should use:

[[Image:Ligationequation.png]]

Using the lengths of sequences we would be ligating, this is the mass that we needed:

X nanograms of insert = [(2)(144bp)(50 ng linearalized plasmid)]/2038bp
X = 7.0657 ng of insert

The following table outlines our calculations for finding the mass of tRNAs we had:

[[Image:Ligationcalculations.png]]

We would attain this mass of tRNA by pipetting an unknown amount of it. In order to attain a more "pipetable" amount of tRNA, we diluted our initial concentration 50X. Since we knew we wanted our final mass to be 7.065 ng, we calculated the volume we would need to take from this new, diluted sample:

DILUTION: 555.48/50 = 11.012
CALCULATION: 11.012 ng/ 1uL = 7.065 ng/ Y uL
Y = .64 uL of insert

Afterwards, Olivia and Alyndria began the actual ligation process while Shamita, Leland, and I filled the wells of the two gels. The first included the two digested reporter parts. We also included a molecular weight marker. Here is a photograph of this gel:

[[Image:2009-06-12 15hr 54min.jpg]]

According to our prior calculations, the Tet plasmid should have been 2958 base pairs long and the RFP plasmid should have been 2380 base pairs long. Our expectations were verified by the data. Shamita and I then sliced the part of thsi gel containing both plasmids and put these slices away to purfiy on Monday.

The second gel contained aliquots of the PCR we had done to attain 10 FML sequences: 5 containing the Tet resistant reporter gene and each of the 5 frameshifts that Davidson was assigned and the other 5 containing the RFP reporter gene and each of the 5 frameshifts as well:

[[Image:2009-06-12 16hr 07min.jpg]]

According to our data, each sequence containing the Tet resistant gene should have been 317 base pairs long and each sequence containing the RFP gene should have been 446 base pairs long. Our data supports our hypothesis, however, the 3rd well was not successful. This well contained the FML consisting of the Tet resistant gene and the frameshift mutant CUAGU. Before leaving work for the day, Shamita and I prepared and performed PCR using two parts containing the tet resistant gene, J31007 and S03710. We wanted to use both to make sure at least one of them works since we could technically use either of them, even though when we first did PCR we only used the S03710 part. Using the known concentrations of each of these parts, and knowing that we needed 1 ng of each part to include in our PCR samples, we calculated the volume of each 100X-diluted solution we would need:

S03710: 1.636 ng/uL = 1 ng/ X uL
X = 0.611 uL

J30017: 0.776 ng/uL = 1 ng/ Y uL
Y = 1.289 uL

We will run another gel with both of these parts on Monday and hopefully at least one of them works.

== [[User:Roclemente|Roclemente]] 17:24, 15 June 2009 (EDT) ==

Each biology member of the Davidson iGEM team has been assigned a 5mer to carry on throughout the rest of their respective processes. I've been assigned the 5mer CUAGU. We verified the PCR of the CUAGU FML containing the RFP gene on Friday. However, we weren't able to verify the CUAGU FML containing the tet resistance gene when we ran it on a gel. Shamita and I redid PCR for two parts containing the tet resistance gene: J31007 and S03710, labeled 2* and 2! respectively. I'm running another gel to verify either or both of them. Both sequences should be 317 bp long (this includes the ATF, logical clause, and the tet resistance gene to the left of the BamH1 restriction site). Here is the gel run we collected. The first column represents the 1 kb DNA marker, and the next two are 2* then 2!:

[[Image:2009-06-15 10hr 34min.jpg]]

The data shows that the PCRs of both parts wound up at the length we expected. The first time we did PCR on J31007, we must have left a solution out of our procedure on accident. I combined both PCRs together since they are the same length and match the length we expected to get.

Next, we cleaned and concentrated our amplified DNA sequence (#D4014 from Zymo Research). I cleaned the two pieces of DNA that contained the 5mer CUAGU. I labeled the tube with DNA also containing the RFP gene as "1" and the tube with DNA also containing the tet resistance gene as "2!". We needed 2 volumes of buffer for every 1 volume of DNA we added. Therefore, for tube "1," I added 95 uL DNA and 190 uL buffer. I added more DNA to tube "2!" since I combined the DNA from two PCR events to one (2* and 2! from earlier).

After cleaning the DNA, we found its concentration using the Nanodrop. In a solution made up of 0.5 uL of the DNA and 2.5 uL of TE, we found the following data from the DNA in this solution:

[[Image:Cleanconcentrate.png]]

The concentration of the DNA we cleaned was the concentration given by the Nanodrop multiplied by 6 (the DNA was dilute 6-fold for the Nanodrop). We used this concentration and the volume of clean DNA we knew we had (9.5 uL) to find the mass of clean DNA we had:

[[Image:Concentrateddna.png]]

Before doing digestion, we used the ligation equation (see "Ligation Protocol" in "Davidson Lab Protocols") to figure out how much mass of the DNA insert we would need later on when we did ligation. For the RFP FML, we found we would want the following mass of insert:

ng of insert = (2)(443)(50)/(2320) = 19.0948 ng

We multiplied this mass by 50 because we're counting on losing DNA between the digestion and cleaning we would need to do leading up to the ligation protocol.

19.0948 ng x 50 = 954.741 ng RFP FML

We found the mass of Tet FML insert we would need for ligation as well:

ng of insert = (2)(314)(50)/(2958) = 10.6153 ng

We multiplied this mass by 50 too:

10.6153 x 50 = 530.764 ng Tet FML

Unfortunately, nobody had enough of their clean DNA to satisfy our careful criteria. However, we would need to do this calculation later on so it was helpful we did it anyway. Instead, we just used all the DNA we had, or 9.5 uL for each reporter gene. Since there were 4 people who needed the same master mix, we made 5 parts of the master mix using the following volumes:

[[Image:Rfptetdigestion.png]]

After realizing that we would have to wait a day for the digestion to take place, we decided to culture every tRNA cell that colonized. The tRNA that we transformed on Friday did not have a good yield, so last night a few people came in and transformed more tRNA cells. We got a few more colonies. To be safe and make sure we cultured each tRNA, we picked every colony visible to us in both the old and new plates. Using our own given tRNAs, I counted the amount of colonies that grew in the plate we transformed on 6/12 and 6/14. There were 4 colonies that grew on both plates so I labeled a tube for each of these. I awas also in charge of the negative control. There were 2 colonies that grew on the negative control plate so I labeled 2 tubes for that. We will be placing them in an incubator overnight.

== [[User:Roclemente|Roclemente]] 12:30, 16 June 2009 (EDT) ==

We checked on the cells we cultured overnight. Of the cells that I cultured (all containing the CUAGU 5mer and the two negative controls), both negative control tubes worked as well as the tubes I labeled 1 (6/14), 5 (6/12), and 7 (6/12). The rest of mine were either red, meaning the plasmids hadn't ligated properly) or the colonies we used were no good. I took these 5 tubes, along with 3 of Olivia's tubes filled with cells that were cultured (tubes 11, 12, and 13) and miniprepped them to isolate the plasmids from the cells. There was no need to colony PCR to screen for successful ligations because this is only used to know which ones to let grow overnight and we had already done this.

As soon as I finished miniprepping them, I had to leave the lab for a little bit, so Olivia took each my miniprep tubes besides the negative controls and Leland took my negative controls. I completed the protocol for digestion of the tRNAs with EcoR1 and Pst1 for Alyndria's tubes when I got back because she had left a little earlier for lunch.

Afterwards, Shamita and I poured a 1.9% concentrated gel to load the digested tRNAs on. We want to verify that these tRNAs are the appropriate size for if they were cut properly. Since we will have to load 37 wells, and the maximum number we can load on one gel is 18 since 2 wells will have to be used by the molecular weight marker, we needed to pour 2 gels and we will also be using a half of a 2.0% agarose gel that was left over from another experiment.

Shamita and I also made new 0.5X running buffer gel. We did this by adding 450 uL buffer and 9 uL ethidium bromide (1 uL EtBr : 50 uL buffer). Alyndria, Leland, and Shamita loaded these gels.

Olivia and I cleaned our PCR DNA using the "Ethanol precipitate DNA (short protocol)" found in the Davidson Lab Protocols link. To begin with, I added 180 uL dH2O to my two samples, CUAGU with RFP and CUAGU with Tet, to bring their volumes up to 200 uL. During one of the last steps of this protocol, when Olivia and I dried out our DNA using the SpeedVac for a period of 10 minutes, the top of my tube containing the CUAGU 5mer and RFP gene broke. I wasn't sure if a pellet was in there, but I still poured out the ethanol and went on with the protocol. At the end, I resuspended what may or may not have been my DNA in 10 uL distilled water. I NanoDropped it afterward and attained the following data concerning both tubes:

[[Image:Failedpcrmeh.png]]

According to this data, any absorption and concentration we may have attained is insignificant because the A-260 must be between 0.05 and 1.50 to be significant and neither of the DNA is. Tomorrow I will do PCR for both FMLs over again.

== [[User:Roclemente|Roclemente]] 14:51, 17 June 2009 (EDT) ==

I found out that my tRNA DNA that I labeled "1" and which contains CUAGU was verified from the gel run Olivia did yesterday (the gel run also included her tRNA DNA samples). At the start of the day I plated this tube of cultured cells again to grow overnight.

Since PCR will take about 3 1/2 hours, I attempted this procedure at the beginning of the day. I plan on making 5 PCR products for each reporter gene since I now know the environmental conditions are correct. I labeled the RFP tubes 1-5 and the Tet tubes 6-10. I first needed to figure out how much of the template DNA I would need in order to have 1 ng template DNA. I made the following calculations to find this out:

[[Image:1ngtemplatedna.png]]

I wanted to add the Monster Mix last, so I made the following table, adding everything else besides the Monster Mix for 5 of the same reporter gene to one tube:

[[Image:Pcrmix.png]]

Unfortunately, when I actually mixed the solution together, I forgot to dilute the template DNA. Therefore, we have 100X more template DNA than we would have had we diluted the template DNA. The PCR can still work, but we will have to constantly remind ourselves that there will be extra template DNA in our solution and that this may skew, however slightly, our results later on.

While I waited for the PCR product, I refocused on getting my tRNA DNA ready for sequencing. Using the NanoDrop, I found the concentration of my miniprepped CUAGU DNA that was verified to be 61 ng/uL. The pre-sequencing protocol calls for 60 ng DNA, so I extracted 1 uL from this tube (the extra DNA will be insignificant in the sequencing procedure). I pipetted this into another tube and used the SpeedVac to dry it out. This is how we will send our DNA to be sequenced.

The following is a summary of steps I put together to help me plan out what I will do after the PCR procedure is over (it will be done at 5 pm today):

1. Combine the five matching tubes into one tube.
2. Run each PCR product on a gel to verify that our expected sequence has been properly amplified.
3. Clean and concentrate plasmids so salt solution is optimal for digestion.
4. Double digest PCR products with EcoR1 and BamH1 or Nco1.
5. Clean and concentrate again to get rid of restriction enzymes.
6. Nanodrop inserts in order to do ligation calculations and verify that sequences are present.
7. Ligate FML inserts with purified plasmids.
8. Transform plasmid into cells.

Since I won't have time to run a gel and take a picture once the PCR procedure is complete (we get out at 5:30 and it will be finished at 5:00), I have begun to prepare the agarose gel for use tomorrow. The size of the band I expect to find for the Tet FML is 349 bp (including the GCAT, BioBrick prefix, ATG, 5mer, and reporter gene until about 20 nucleotides or so past the restriction site we will cut at) and 474 bp for the RFP FML. According to the "Optimize Your Gel" link on the Davidson Lab Protocols page, a 1.6% gel will optimize how well I see my Tet bands and a 1.3% gel will optimize how well I see my RFP bands. I can use a gel with a concentration in between both of the optimal concentrations for Tet and RFP so that I can use one gel for both. Therefore, I will use a 1.45% gel. 1.45% of 60 uL buffer solution tells me that I should measure out 0.87 g agar for this gel.

== [[User:Roclemente|Roclemente]] 14:00, 18 June 2009 (EDT) ==

I checked on my PCR products this morning. Tubes 1 and 2 (both containing the RFP FML) had solution scattered on the tube's cover and walls so I placed them in the centrifuge for a bit. Unfortunately, I forgot to put these 500 uL tubes in holders when I centrifuged them so these tubes were destroyed. This was okay though because I was just going to combine each respective FML into one tube. I would have 300 uL of RFP FML PCR product instead of 500 uL and I put all of this in tube 5. I still had 500 uL of Tet FML PCR product left and put all of this in tube 10.

I ran 5 uL of both RFP and Tet FMLs on a gel, as well as a 1 kb molecular weight marker. The MW marker is in lane 1, the RFP is in lane 3, and the Tet is in lane 4 (Leland ran his PCR product in lane 2):

[[Image:2009-06-18 09hr 56min.jpg]]

I was expecting the RFP PCR product to be 474 bp long and the Tet PCR product to be 349 bp long. Both appear to come out a bit shorter, but Dr. Campbell and I agreed that we wouldn't count these sequences out because the bands were quite close to what we expected.

I proceeded to clean and concentrate these two sequences using the procedure outlined in the "Clean and Concentrate DNA (after PCR, before digestion)." Now I had 295 uL of the Tet sequence and 495 uL of the RFP sequence. Since the procedure says I can only have 800 uL in the column at a time, with DNA:Buffer at a ratio of 1:2, I mixed the two in a column in a series of two rounds using the following volumes:

[[Image:Clean.png]]

I Nanodropped my DNA after cleaning it and attained the following results:

[[Image:Nanome.png]]

I wanted the OD260 (Optical Density at 260) to be under 1.5, which it is for both. I lost quite a bit of Tet DNA in the process, and will only continue to lose DNA. To account for this loss of DNA, Dr. Campbell suggested it would be a good idea to heat inactivate the restriction enzymes to get rid of them after digestion instead of cleaning and concentrating the DNA over again.

I double digested both RFP and Tet FML sequences. I used all 9.5 uL of my DNA. The following table outlines how much volume of each material I mixed together:

[[Image:2nddoubledigest.png]]

While I waited 3 hours for my digestion to take place, I prepared 6 tubes to make more liquid cultures of the RFP and Tet vectors to grow more receiving vectors. 3 tubes would be to make RFP cultures and the other 3 would be for the Tet cultures. For RFP, I used the part I715039 and for Tet, I used the part J31007.

Once the digestion took place, Dr. Campbell notified me that heat inactivation was capable for Nco1 but not BamH1. There were 3 options for me to take in order to clean the Tet FML, the protocols for all of which can be found on the Davidson Lab Protocols link:

# [http://www.bio.davidson.edu/courses/Molbio/Protocols/Clean_Concentrate.html Clean and Concentrate DNA (after PCR, before digestion)]
# [http://www.bio.davidson.edu/courses/Molbio/Protocols/clean_short.html Ethanol Precipitate DNA (short protocol)]

IGEM 2009 notebook

2009-06-18T20:37:31Z

ShPunjabi: /* Roclemente 14:00, 18 June 2009 (EDT) */

== [[User:Roclemente|Roclemente]] 16:54, 3 June 2009 (EDT) ==

Shamita and I are trying to find suitable reporter proteins to use. Yesterday, Leland and Alyndria were working on ways to insert the gene sequences into the plasmid. Upon seeing how they wanted to manipulated the reporter gene to include the logical clauses, we came up with a few criteria for the reporter genes we would use. The following criteria for genes are listed in order of the broadest aspect to look at to the narrowest aspect:

a) Doesn't contain restriction sites for the 4 restriction enzymes (EcoR1, Xbal, Spel, Pst1) used to cleave the Biobrick part out of the plasmid.

b) Contains 6 cutter restriction sites.

c) These restriction enzymes aren't blunt (cleave straight down at one spot).

d) These restriction sites are close to thge 5' (beginning) end of the sequence.

e) These enzymes are easiest to work with and cheapest.

We are finding the part numbers of the reporter genes we want to use (antibiotic resistance, fluorescence, ''LacZ'') through our own GCAT because we know these ones work. We are then locating these parts on the parts registry [http://www.partsregistry.org] website. We copied and pasted the gene sequences we obtained from the registry onto the ApE software [http://www.biology.utah.edu/jorgensen/wayned/ape/]. From here, we were able to generate a genetic map of each gene that outlined each restriction site that fit our criteria. We put each genetic map, alongside the part number used, into a Word document:

http://gcat.davidson.edu/GcatWiki/images/0/0e/Restriction_Site_Mapping_on_Reporter_Genes.doc

So it looks like we've changed directions. The team is looking towards the wet lab portion of our research more now. Instead of simply speculating about which reporter proteins we think will work best and which tRNA suppressors to use, we're going to physically test it out ourselves. A few tasks at hand in the second part of this afternoon:

1. What are the DNA gene sequences of the 12 different tRNA suppressors we want to use?

2. What is the sequence of the DNA that is cleaved off of both ends of the tRNA when it is transcribed?

We e-mailed Anderson to find out the exact tRNA gene sequences because we had a hard time finding this information everywhere else. Most papers we found describing the 5-base codon tRNA suppressors simply referred to Anderson's paper [http://gcat.davidson.edu/GcatWiki/images/6/6f/AndersonSchultz2002ChemBiol.pdf]. Anderson replied near the end of the day with the gene sequence. According to his email, the only difference between different tRNA genes is the anticodon loop. Therefore, if we just substitute all the anticodon loops in the invariable part of the sequence, we have our different tRNA sequences. We plan on using the Lancilator to help us break these tRNA sequences into smaller oligonucleotides that can anneal at around the same temperature because it is not possible for us to order oligos that are greater than 70 nucleotides long.

== [[User:Roclemente|Roclemente]] 17:21, 4 June 2009 (EDT) ==

I started out the day looking at my time-sensitive group research qustion about reporter proteins. I started making a table listing the advantages and disadvantages of different reporter proteins. Then the group found out that there was a major roadblock to our project: a stop codon was located in our ATG-5mer oligo. The stop codon would have been part of the BioBrick scar. The group looked at several ways to work around this problem. The idea that I looked most into was replacing the restriction sites that were used in the scar through standard assembly. According to the judging criteria for iGEM, I found that a team could possibly alter the standard assembly method as long as they wrote a letter to the iGEM judges explaining our plan in a detailed manner. From there, I set off to understand the BioBrick scar more by making a document highlighting the exact positions of the rsetriction sites on the BioBrick parts and how they were used to put more than one part together. I then found several other pairs of restriction sites that complemented one another and could be used in place of the standard scar between Xba1 and Spe1. At the end of the day, the group got the chance to speak with Dr. Campbell and he suggested a hybrid idea which would combine Davidson's restriction site idea with Missouri's PCR idea. I'm not quite sure of what this idea is exactly yet, but we'll all clarify our vague idea of it tomorrow morning after a talk with Dr. Eckdahl.

== [[User:Roclemente|Roclemente]] 17:13, 5 June 2009 (EDT) ==

This morning we began exploring different options for bypassing the stop codon problem from yesterday. If we wanted to make sure we were using the BioBrick parts in our GCatalog, there was no way we were going to be getting rid of the Xba1 restriction site that contained the "TAG" which codes for a stop codon. Therefore, we proposed looking at restriction sites within the reporter genes instead, therefore addressing the already-placed BioBrick ends. Using the document me and Shamita created on "Restriction Site Mapping on Reporter Genes" (see first journal entry), we determined that the reporter gene with the restriction site closest to the beginning of the sequence was YFP. We proposed cutting the YFP gene that we already had in stock at this restriction site and inserting an oligo (which we would have ordered) with a sequence in the following order: "BioBrick prefix - ATG - 5mer - part of YFP gene to the left of the cleaved restriction site."

After clarifying our own idea with one another, we spoke with some Missouri kids to clarify the said "hybrid" idea which, according to Dr. Campbell, combined the ideas of both campuses. The hybrid idea was based upon our idea of cutting the reporter gene at a restriction site as well, except that our primer would start out with a primer for our reporter gene, and to the left of the primer would be a "tail" consisting of the ATG and 5mer and either an Xba1 or EcoR1 restriction site (We still have to decide this. Our decision will probably be based on the restriction site we end up using in our reporter gene and whether or not that is complementary to either the Xba1 or EcoR1 sites.). We would use the PCR technique and use our in-stock reporter gene strand as our template strand (it will be denatured into single strands) and we will add our oligo. We propose that the primer will synthesize the rest of the YFP strand as well as add on nucleotides to complete the oligo tail. In the end, we will have a double stranded DNA sequence consisting of either the Xba1 or EcoR1 site, the ATG, the 5mer, and the reporter gene. The beauty of this hybrid idea is that we have flexibility as far as the reporter gene we choose to use since all we really need for it is the beginning sequence (for the primer) and a known, "hearty" restriction site. Once we cut this double strand at this restriction site on the reporter gene as well as at the Xba1 or EcoR1 sites, we can insert this into a plasmid that contains the rest of that reporter gene after the restriction site we choose to use and the complementary Xba1 or EcoR1 site.

I made a Word document outlining the promoter and RBS parts we have in our registry on the Davidson campus. Leland and I went through the registry parts on the GCATalog to look for potential promoters and RBS parts. We went through the document as a group and analyzed each of these potential parts on partsregistry.org to see which ones would most likely work the best in the miniprep we'll start preparing for on Sunday evening. Here is the document we wound up with:

http://gcat.davidson.edu/GcatWiki/images/d/d2/Promoters_and_RBS.doc

== [[User:Roclemente|Roclemente]] 10:40, 8 June 2009 (EDT) ==

We're starting the day by finalizing the oligos we will be ordering to have synthesized. Two groups of oligos must be sequenced: primers and tRNA gene sequence. The primers will consist of a forward and reverse primer. The forward primer will consist of the following in this 5' - 3' order: GCAT - BioBrick Prefix - ATF - 5mer - first 20 nucleotides in the reporter gene sequence. These 20 nucleotides won't include the ATG that naturally comes at the beginning of the reporter gene because we want to make sure the ribosome attaches to the first ATG and reads the logical clause instead of going straight to the ATG at the start of the reporter gene. The reverse primer will consist of the first 20 nucleotides downstream of the restriction site we plan to use in the reporter gene. I worked on finding the primers for the Tet resistance gene (part J31007) while Lyn worked on finding the primers for the RGP gene. Dr. Campbell decided we should use the BamH1 restriction enzyme because it is very "hardy." The following document outlines the 5 forward primers (each with one of the five variable 5mers) we will order for amplification of the Tet resistance FML:

[[Image:Tet resistance forward primers.png]]

This is the reverse primer we will order for the Tet resistance FML:

[[Image:Tet resistsance reverse primer.png]]

== [[User:Roclemente|Roclemente]] 11:54, 9 June 2009 (EDT) ==

We need to digest the plasmids at the EcoR1 and Pst siets. Everytime we're going to set up a digestion, we'll want the following in these volumes: 10x Buffer (2 uL), DNA (~ 3 uL), enzymes 1 and 2 (1 uL each), water (13 uL), and final volume of all of these put together should be 20 uL.

Some notes about digestion: Enzymes should make up 10% of the final volume. As a general rule of thumb, make sure the tip of your pipet is close to the surface of the solution. This is especially pertinent when gathering 1 microL of each of the enzymes. The most sensitive components are the enzymes, so they should go last.

We'll have a bunch of tubes with each 3 microL of the specific DNA. We'll make a cocktail in a bigger tube consisting of the buffer, 2 enzymes, and water. The rationale behind thissis that you'll be less likely to make a bigger mistake with a bigger volume (versus getting 1 microL of enzyme per specific DNA). You'll also know that there is no problem with the enzyme if all the DNA don't cut. If only one specific DNA doesn't work, then we'll know that it's because there's something wrong with the specific DNA rather than anything in the "Master Mix." Since we have to do 11 digestions, we'll make enough of the master mix for 12 tubes to make sure we have enough. We'll take 17 microL of the Master Mix to each tube of DNA.

The optimal temperature for most enzymes is 37 degreees celsius. You'd like to have enzymes with the same optimal temperatures (for ex. restriction enzymes that start with B usually have an optimal temperature near 50 degrees. You wouldn't want to use both B-enzyme and EcoR1 because EcoR1 would die).

'''What percent gel should we use?'''

Look at the Davidson lab protocol on the Davidson-Missouri Wiki. Generally, bigger molecules require an agarose gel with a lower concentration.

'''What's next in the future?'''

5' end additions are done by PCR. We'll ampliffy this double-stranded DNA. We'll cut with both EcoR1 and BamH1 or Nco1 (depending on which reporter gene you're using). Then we'll cut the plasmid with the reporter with the same restriction enzymes used.Then, to throw away the remaining DNA that we don't want, we'll run a gel and throw away the smaller band (because the larger band is probably made of the plasmid we want to keep).Then we'll take the double stranded DNA we amplified and the purified plasmid and ligate, transform, and screen them and verify the sequence.

While we wait for the oligos, we can cut the plasmid with reporter on the same restriction enzyme already.

'''How are we building the tRNAs?'''

We'll assemble the oligos into genes by mixing our oligos together, boiling them, and very slowly allowing them to cool. This will require many calculations. We'll cut the plasmids with EcoR1 and Pst1. to be ready to receive the enes we're assembling.We'll have to gel purify the plasmids to get them away from their inserts. We'll ligate, transform, and screen the genes and the plasmids and verify the sequence. On paper you shouldn't have two sites cut with different restriction enzymes stick together, but if there's selection pressure, these sites may cut some ends to fit together. This is why we must screen them.

After Dr. Campbell taught us all of the above, the 5 biologists put together a document outlining exactly how we would go about cutting the two sets of plasmids (one set will be cut with Eco R1 and Pst1 and othe other set will be cut with Eco R1 and BamH1 or Nco1). The following table outlines which biologist was going to work with which sample of DNA:

[[Image:Names and DNA.jpg]]

The following summarize the diferent materials and volumes of these materials that we'll need for our digestion of the plasmids:

[[Image:Digestion materials.png]]

This last table highlights the agarose percent concentration we should use for each part according to how many base pairs it contains:

[[Image:Screen-capture-3.png]]

The biologists digested the plasmids that we plan on using once our oligos arrive in a day or two. Afterwards, we made the two agarose gels that were most compatible with the parts we were going to run. This gel is going to purify the plasmids so that we have the parts that we're going to use later on, minus the rest of the DNA in the plasmid that we don't need. Before physically creating the gel, we figured out how much agarose we would need to make our two different agarose gel concentrations, 0.8% and 2.5%. Next, we made sure the mold and the big teeth were in place. We used the big teeth at the end of the mold because we plan on running 20 uL of DNA versus a running a smaller portion which would require us to use small teeth. We then measured the amount of agarose that we calculated we would need, placed this in a volumetric flask greater than 60 uL to allow for fizz to rise up, then measured out 60 uL of buffer and mixed this in the same flask with the agarose. We placed clear plastic wrap on top of the flask and heated it up for 1 minute and 20 seconds in the microwave. After we heated it, we took it out and checked to see if there was still agarose present that hadn't yet melted. There was, so we placed it back in the microwave and heated it for another 20 seconds. All the agarose had melted. We added 1 uL of EtBr (ethidium bromide) to the solution and quickly poured this into the mold. It needs about 15 to 20 minutes to cool. Dr. Campbell made the agarose gel that was 2.5% concentrated gel while we watched. We then made the 0.8% concentrated gel while he watched.

The following table lists the order in which we loaded our DNA into the gel. The first column all the way to the left is represented by number 1:

[[Image:Screen-capture-2.png]]

== [[User:Roclemente|Roclemente]] 08:46, 10 June 2009 (EDT) ==

After about 30 minutes after we began the gel electrophoresis, we looked at our gels with UV light to see how far the inserts had travelled and where the plasmids were. Then we took each gel to be photographed in the freezer room (231). We used the following DNA length marker to measure how long each sequence represented by each band was:

[[Image:Screen-capture-1.png]]

We compared our data to the lengths we expected to get according to the registry online. The first gel we photographed was the one that was 2.5% concentrated:

[[Image:2009-06-09 14hr 44min.jpg]]

The first two columns of the DNA we ran, both consisting of B0300, were fairly the same as the length we expected. However, the last two columns, consisting of K091111 and K091112 respectively were supposed to be 86 bp long, however, according to our data, K091112 was longer. Leland and I looked at Pallavi Penumetcha's paper which delved more closely into these two parts and what she wrote in her paper and what she entered into the registry conflict. We are currently corresponding with her to figure out the exact discrepancy.

We then photographed the 0.8% gel:

[[Image:2009-06-09 15hr 21min.jpg]]

The length of the sequences in almost every column matched up with the lengths we expected. Only the 4th and 5th columns didn't come out clearly. We think that the DNA inserted in column 5 weren't inserted properly and diffused on its own. We couldn't see bands in the 4th column at all until we adjusted the color a bit on the photographer and saw that column 4 was pretty much identical to column 5. We assumed that column 5 bled into column 4.

== [[User:Roclemente|Roclemente]] 15:14, 11 June 2009 (EDT) ==

We are digesting two parts today: I715039 (pLac+RBS+RFP) and S03710 (Tet Resistance). We want both the plasmids and the inserts in each case. We are aiming for a final volume of 40 uL for each part. This means that each digestion will consist of 4 uL buffer, 2 uL EcoR1, and 2 uL of their other respective restriction site (BamH1 for S03710 and Nco1 for I715039). The only parts that will vary are the amount of DNA and water we use. The amount of DNA we use depends on the concentration of the DNA we have (found through use of the NanoDrop). Since we want 2500 ng of DNA, we can use the concentration of DNA to figure out how much volume of this concentrated DNA we'll want to use in your digestion. If we subtract the volumes of buffer, two restriction enzymes, and DNA we plan on using from 40 uL, we'll have the amount of water we need to add. Here are the different volumes of materials we plan on using in our double digestion:

[[Image:DoubledigestRFPTetR.png]]

Now that we know how much of each material we were going to use, we needed to find out which buffer would be compatible with each sample according to the restriction enzymes we were using and how effective they'd be in different salt solutions. Online on the Promega "Compatible Buffers" website, we were able to find which Promega buffers would be most compatible when doing a double digest with EcoR1 and BamH1:

[[Image:BamEcoR1.png]]

Here is a chart noting buffers and their compatiblity with EcoR1 and Nco1:

[[Image:NcoEcoR1.png]]

We still have to wait for the restriction enzymes to arrive, so afterwards, we transformed three different inserts containing needed promoters and RBSs into vectors. We used the Zippy Transformation method. These promoters and RBSs will later be added to the FMLs once they are already inserted into the reporter cells. The following table outlines these inserts and part numbers:

[[Image:Promoterinsertspartnumbers.png]]

The restriction enzymes have arrived. We will now be able to do PCR to amplify the RFP gene (I715039) and the Tet resistance gene (S03710) as templates. We will be using the primers which have also arrived. We ordered forward and reverse primers so that our amplified sequence will consist of the following in this order:

GCAT-ATG-5mer-Reporter Gene

The following table lists the materials will be needed for this PCR technique which uses the "Green Promega master (Monster) Mix:"

[[Image:Greenpromegamastermix.png]]

This is the PCR procedure:

[[Image:Temperaturecycle.png]]

These are the variables that we will be adding to each separate sequence we want to be amplified:

[[Image:Tobeamplified.png]]

This table shows how we labeled the tubes based on how we divided the PCR work for the 10 tubes:

[[Image:Pcrtubes.png]]

Now we are giong to assemble the tRNA oligos together to form the tRNA genes. We could only order a certain length of sequence and the tRNA genes outdid these specified lengths. We used the Lancilator to find various oligonucleotides that overlapped and would be able to anneal together to form the tRNA genes. We used the Davidson lab protocol entitled "Buiding dsDNA with Oligos" to assemble the oligos:

[[Image:Oligoassembly.png]]

== [[User:Roclemente|Roclemente]] 22:46, 12 June 2009 (EDT) ==

We had two main goals for today:
1. perform a tRNA ligation and transformation
2. perform two electrophoreses: one to verify the PCR sequences we obtained and another to purify the digested reporter plasmids we would need

We began the day by pouring the agarose gels according to which concentration of buffer would be most compatible with the molecular weight of the substance we wanted to isolate. For both the digestion of E01010 and J31007, according to the "Optimize your Gel" website found on the Davidson Lab Protocols site for iGEM 2009, we wanted a 0.4% concentrated gel. For the PCRs, we wanted a 1.6% concentrated gel to optimize our retrieval of the Tet gene and a 1.4% concentrated gel to optimize our retrieval of the RFP gene.

While we waited for the gels to harden for about 30 minutes, we began calculating how much of each material we would need to ligate our tRNAs. We used the following equation to find out how much insert we should use:

[[Image:Ligationequation.png]]

Using the lengths of sequences we would be ligating, this is the mass that we needed:

X nanograms of insert = [(2)(144bp)(50 ng linearalized plasmid)]/2038bp
X = 7.0657 ng of insert

The following table outlines our calculations for finding the mass of tRNAs we had:

[[Image:Ligationcalculations.png]]

We would attain this mass of tRNA by pipetting an unknown amount of it. In order to attain a more "pipetable" amount of tRNA, we diluted our initial concentration 50X. Since we knew we wanted our final mass to be 7.065 ng, we calculated the volume we would need to take from this new, diluted sample:

DILUTION: 555.48/50 = 11.012
CALCULATION: 11.012 ng/ 1uL = 7.065 ng/ Y uL
Y = .64 uL of insert

Afterwards, Olivia and Alyndria began the actual ligation process while Shamita, Leland, and I filled the wells of the two gels. The first included the two digested reporter parts. We also included a molecular weight marker. Here is a photograph of this gel:

[[Image:2009-06-12 15hr 54min.jpg]]

According to our prior calculations, the Tet plasmid should have been 2958 base pairs long and the RFP plasmid should have been 2380 base pairs long. Our expectations were verified by the data. Shamita and I then sliced the part of thsi gel containing both plasmids and put these slices away to purfiy on Monday.

The second gel contained aliquots of the PCR we had done to attain 10 FML sequences: 5 containing the Tet resistant reporter gene and each of the 5 frameshifts that Davidson was assigned and the other 5 containing the RFP reporter gene and each of the 5 frameshifts as well:

[[Image:2009-06-12 16hr 07min.jpg]]

According to our data, each sequence containing the Tet resistant gene should have been 317 base pairs long and each sequence containing the RFP gene should have been 446 base pairs long. Our data supports our hypothesis, however, the 3rd well was not successful. This well contained the FML consisting of the Tet resistant gene and the frameshift mutant CUAGU. Before leaving work for the day, Shamita and I prepared and performed PCR using two parts containing the tet resistant gene, J31007 and S03710. We wanted to use both to make sure at least one of them works since we could technically use either of them, even though when we first did PCR we only used the S03710 part. Using the known concentrations of each of these parts, and knowing that we needed 1 ng of each part to include in our PCR samples, we calculated the volume of each 100X-diluted solution we would need:

S03710: 1.636 ng/uL = 1 ng/ X uL
X = 0.611 uL

J30017: 0.776 ng/uL = 1 ng/ Y uL
Y = 1.289 uL

We will run another gel with both of these parts on Monday and hopefully at least one of them works.

== [[User:Roclemente|Roclemente]] 17:24, 15 June 2009 (EDT) ==

Each biology member of the Davidson iGEM team has been assigned a 5mer to carry on throughout the rest of their respective processes. I've been assigned the 5mer CUAGU. We verified the PCR of the CUAGU FML containing the RFP gene on Friday. However, we weren't able to verify the CUAGU FML containing the tet resistance gene when we ran it on a gel. Shamita and I redid PCR for two parts containing the tet resistance gene: J31007 and S03710, labeled 2* and 2! respectively. I'm running another gel to verify either or both of them. Both sequences should be 317 bp long (this includes the ATF, logical clause, and the tet resistance gene to the left of the BamH1 restriction site). Here is the gel run we collected. The first column represents the 1 kb DNA marker, and the next two are 2* then 2!:

[[Image:2009-06-15 10hr 34min.jpg]]

The data shows that the PCRs of both parts wound up at the length we expected. The first time we did PCR on J31007, we must have left a solution out of our procedure on accident. I combined both PCRs together since they are the same length and match the length we expected to get.

Next, we cleaned and concentrated our amplified DNA sequence (#D4014 from Zymo Research). I cleaned the two pieces of DNA that contained the 5mer CUAGU. I labeled the tube with DNA also containing the RFP gene as "1" and the tube with DNA also containing the tet resistance gene as "2!". We needed 2 volumes of buffer for every 1 volume of DNA we added. Therefore, for tube "1," I added 95 uL DNA and 190 uL buffer. I added more DNA to tube "2!" since I combined the DNA from two PCR events to one (2* and 2! from earlier).

After cleaning the DNA, we found its concentration using the Nanodrop. In a solution made up of 0.5 uL of the DNA and 2.5 uL of TE, we found the following data from the DNA in this solution:

[[Image:Cleanconcentrate.png]]

The concentration of the DNA we cleaned was the concentration given by the Nanodrop multiplied by 6 (the DNA was dilute 6-fold for the Nanodrop). We used this concentration and the volume of clean DNA we knew we had (9.5 uL) to find the mass of clean DNA we had:

[[Image:Concentrateddna.png]]

Before doing digestion, we used the ligation equation (see "Ligation Protocol" in "Davidson Lab Protocols") to figure out how much mass of the DNA insert we would need later on when we did ligation. For the RFP FML, we found we would want the following mass of insert:

ng of insert = (2)(443)(50)/(2320) = 19.0948 ng

We multiplied this mass by 50 because we're counting on losing DNA between the digestion and cleaning we would need to do leading up to the ligation protocol.

19.0948 ng x 50 = 954.741 ng RFP FML

We found the mass of Tet FML insert we would need for ligation as well:

ng of insert = (2)(314)(50)/(2958) = 10.6153 ng

We multiplied this mass by 50 too:

10.6153 x 50 = 530.764 ng Tet FML

Unfortunately, nobody had enough of their clean DNA to satisfy our careful criteria. However, we would need to do this calculation later on so it was helpful we did it anyway. Instead, we just used all the DNA we had, or 9.5 uL for each reporter gene. Since there were 4 people who needed the same master mix, we made 5 parts of the master mix using the following volumes:

[[Image:Rfptetdigestion.png]]

After realizing that we would have to wait a day for the digestion to take place, we decided to culture every tRNA cell that colonized. The tRNA that we transformed on Friday did not have a good yield, so last night a few people came in and transformed more tRNA cells. We got a few more colonies. To be safe and make sure we cultured each tRNA, we picked every colony visible to us in both the old and new plates. Using our own given tRNAs, I counted the amount of colonies that grew in the plate we transformed on 6/12 and 6/14. There were 4 colonies that grew on both plates so I labeled a tube for each of these. I awas also in charge of the negative control. There were 2 colonies that grew on the negative control plate so I labeled 2 tubes for that. We will be placing them in an incubator overnight.

== [[User:Roclemente|Roclemente]] 12:30, 16 June 2009 (EDT) ==

We checked on the cells we cultured overnight. Of the cells that I cultured (all containing the CUAGU 5mer and the two negative controls), both negative control tubes worked as well as the tubes I labeled 1 (6/14), 5 (6/12), and 7 (6/12). The rest of mine were either red, meaning the plasmids hadn't ligated properly) or the colonies we used were no good. I took these 5 tubes, along with 3 of Olivia's tubes filled with cells that were cultured (tubes 11, 12, and 13) and miniprepped them to isolate the plasmids from the cells. There was no need to colony PCR to screen for successful ligations because this is only used to know which ones to let grow overnight and we had already done this.

As soon as I finished miniprepping them, I had to leave the lab for a little bit, so Olivia took each my miniprep tubes besides the negative controls and Leland took my negative controls. I completed the protocol for digestion of the tRNAs with EcoR1 and Pst1 for Alyndria's tubes when I got back because she had left a little earlier for lunch.

Afterwards, Shamita and I poured a 1.9% concentrated gel to load the digested tRNAs on. We want to verify that these tRNAs are the appropriate size for if they were cut properly. Since we will have to load 37 wells, and the maximum number we can load on one gel is 18 since 2 wells will have to be used by the molecular weight marker, we needed to pour 2 gels and we will also be using a half of a 2.0% agarose gel that was left over from another experiment.

Shamita and I also made new 0.5X running buffer gel. We did this by adding 450 uL buffer and 9 uL ethidium bromide (1 uL EtBr : 50 uL buffer). Alyndria, Leland, and Shamita loaded these gels.

Olivia and I cleaned our PCR DNA using the "Ethanol precipitate DNA (short protocol)" found in the Davidson Lab Protocols link. To begin with, I added 180 uL dH2O to my two samples, CUAGU with RFP and CUAGU with Tet, to bring their volumes up to 200 uL. During one of the last steps of this protocol, when Olivia and I dried out our DNA using the SpeedVac for a period of 10 minutes, the top of my tube containing the CUAGU 5mer and RFP gene broke. I wasn't sure if a pellet was in there, but I still poured out the ethanol and went on with the protocol. At the end, I resuspended what may or may not have been my DNA in 10 uL distilled water. I NanoDropped it afterward and attained the following data concerning both tubes:

[[Image:Failedpcrmeh.png]]

According to this data, any absorption and concentration we may have attained is insignificant because the A-260 must be between 0.05 and 1.50 to be significant and neither of the DNA is. Tomorrow I will do PCR for both FMLs over again.

== [[User:Roclemente|Roclemente]] 14:51, 17 June 2009 (EDT) ==

I found out that my tRNA DNA that I labeled "1" and which contains CUAGU was verified from the gel run Olivia did yesterday (the gel run also included her tRNA DNA samples). At the start of the day I plated this tube of cultured cells again to grow overnight.

Since PCR will take about 3 1/2 hours, I attempted this procedure at the beginning of the day. I plan on making 5 PCR products for each reporter gene since I now know the environmental conditions are correct. I labeled the RFP tubes 1-5 and the Tet tubes 6-10. I first needed to figure out how much of the template DNA I would need in order to have 1 ng template DNA. I made the following calculations to find this out:

[[Image:1ngtemplatedna.png]]

I wanted to add the Monster Mix last, so I made the following table, adding everything else besides the Monster Mix for 5 of the same reporter gene to one tube:

[[Image:Pcrmix.png]]

Unfortunately, when I actually mixed the solution together, I forgot to dilute the template DNA. Therefore, we have 100X more template DNA than we would have had we diluted the template DNA. The PCR can still work, but we will have to constantly remind ourselves that there will be extra template DNA in our solution and that this may skew, however slightly, our results later on.

While I waited for the PCR product, I refocused on getting my tRNA DNA ready for sequencing. Using the NanoDrop, I found the concentration of my miniprepped CUAGU DNA that was verified to be 61 ng/uL. The pre-sequencing protocol calls for 60 ng DNA, so I extracted 1 uL from this tube (the extra DNA will be insignificant in the sequencing procedure). I pipetted this into another tube and used the SpeedVac to dry it out. This is how we will send our DNA to be sequenced.

The following is a summary of steps I put together to help me plan out what I will do after the PCR procedure is over (it will be done at 5 pm today):

1. Combine the five matching tubes into one tube.
2. Run each PCR product on a gel to verify that our expected sequence has been properly amplified.
3. Clean and concentrate plasmids so salt solution is optimal for digestion.
4. Double digest PCR products with EcoR1 and BamH1 or Nco1.
5. Clean and concentrate again to get rid of restriction enzymes.
6. Nanodrop inserts in order to do ligation calculations and verify that sequences are present.
7. Ligate FML inserts with purified plasmids.
8. Transform plasmid into cells.

Since I won't have time to run a gel and take a picture once the PCR procedure is complete (we get out at 5:30 and it will be finished at 5:00), I have begun to prepare the agarose gel for use tomorrow. The size of the band I expect to find for the Tet FML is 349 bp (including the GCAT, BioBrick prefix, ATG, 5mer, and reporter gene until about 20 nucleotides or so past the restriction site we will cut at) and 474 bp for the RFP FML. According to the "Optimize Your Gel" link on the Davidson Lab Protocols page, a 1.6% gel will optimize how well I see my Tet bands and a 1.3% gel will optimize how well I see my RFP bands. I can use a gel with a concentration in between both of the optimal concentrations for Tet and RFP so that I can use one gel for both. Therefore, I will use a 1.45% gel. 1.45% of 60 uL buffer solution tells me that I should measure out 0.87 g agar for this gel.

== [[User:Roclemente|Roclemente]] 14:00, 18 June 2009 (EDT) ==

I checked on my PCR products this morning. Tubes 1 and 2 (both containing the RFP FML) had solution scattered on the tube's cover and walls so I placed them in the centrifuge for a bit. Unfortunately, I forgot to put these 500 uL tubes in holders when I centrifuged them so these tubes were destroyed. This was okay though because I was just going to combine each respective FML into one tube. I would have 300 uL of RFP FML PCR product instead of 500 uL and I put all of this in tube 5. I still had 500 uL of Tet FML PCR product left and put all of this in tube 10.

I ran 5 uL of both RFP and Tet FMLs on a gel, as well as a 1 kb molecular weight marker. The MW marker is in lane 1, the RFP is in lane 3, and the Tet is in lane 4 (Leland ran his PCR product in lane 2):

[[Image:2009-06-18 09hr 56min.jpg]]

I was expecting the RFP PCR product to be 474 bp long and the Tet PCR product to be 349 bp long. Both appear to come out a bit shorter, but Dr. Campbell and I agreed that we wouldn't count these sequences out because the bands were quite close to what we expected.

I proceeded to clean and concentrate these two sequences using the procedure outlined in the "Clean and Concentrate DNA (after PCR, before digestion)." Now I had 295 uL of the Tet sequence and 495 uL of the RFP sequence. Since the procedure says I can only have 800 uL in the column at a time, with DNA:Buffer at a ratio of 1:2, I mixed the two in a column in a series of two rounds using the following volumes:

[[Image:Clean.png]]

I Nanodropped my DNA after cleaning it and attained the following results:

[[Image:Nanome.png]]

I wanted the OD260 (Optical Density at 260) to be under 1.5, which it is for both. I lost quite a bit of Tet DNA in the process, and will only continue to lose DNA. To account for this loss of DNA, Dr. Campbell suggested it would be a good idea to heat inactivate the restriction enzymes to get rid of them after digestion instead of cleaning and concentrating the DNA over again.

I double digested both RFP and Tet FML sequences. I used all 9.5 uL of my DNA. The following table outlines how much volume of each material I mixed together:

[[Image:2nddoubledigest.png]]

While I waited 3 hours for my digestion to take place, I prepared 6 tubes to make more liquid cultures of the RFP and Tet vectors to grow more receiving vectors. 3 tubes would be to make RFP cultures and the other 3 would be for the Tet cultures. For RFP, I used the part I715039 and for Tet, I used the part J31007.

Once the digestion took place, Dr. Campbell notified me that heat inactivation was capable for Nco1 but not BamH1. There were 3 options for me to take in order to clean the Tet FML, the protocols for all of which can be found on the Davidson Lab Protocols link:

a) [[http://www.bio.davidson.edu/courses/Molbio/Protocols/Clean_Concentrate.html Clean and Concentrate DNA (after PCR, before digestion)]]

IGEM 2009 notebook

2009-06-18T20:36:13Z

ShPunjabi: /* Roclemente 14:00, 18 June 2009 (EDT) */

== [[User:Roclemente|Roclemente]] 16:54, 3 June 2009 (EDT) ==

Shamita and I are trying to find suitable reporter proteins to use. Yesterday, Leland and Alyndria were working on ways to insert the gene sequences into the plasmid. Upon seeing how they wanted to manipulated the reporter gene to include the logical clauses, we came up with a few criteria for the reporter genes we would use. The following criteria for genes are listed in order of the broadest aspect to look at to the narrowest aspect:

a) Doesn't contain restriction sites for the 4 restriction enzymes (EcoR1, Xbal, Spel, Pst1) used to cleave the Biobrick part out of the plasmid.

b) Contains 6 cutter restriction sites.

c) These restriction enzymes aren't blunt (cleave straight down at one spot).

d) These restriction sites are close to thge 5' (beginning) end of the sequence.

e) These enzymes are easiest to work with and cheapest.

We are finding the part numbers of the reporter genes we want to use (antibiotic resistance, fluorescence, ''LacZ'') through our own GCAT because we know these ones work. We are then locating these parts on the parts registry [http://www.partsregistry.org] website. We copied and pasted the gene sequences we obtained from the registry onto the ApE software [http://www.biology.utah.edu/jorgensen/wayned/ape/]. From here, we were able to generate a genetic map of each gene that outlined each restriction site that fit our criteria. We put each genetic map, alongside the part number used, into a Word document:

http://gcat.davidson.edu/GcatWiki/images/0/0e/Restriction_Site_Mapping_on_Reporter_Genes.doc

So it looks like we've changed directions. The team is looking towards the wet lab portion of our research more now. Instead of simply speculating about which reporter proteins we think will work best and which tRNA suppressors to use, we're going to physically test it out ourselves. A few tasks at hand in the second part of this afternoon:

1. What are the DNA gene sequences of the 12 different tRNA suppressors we want to use?

2. What is the sequence of the DNA that is cleaved off of both ends of the tRNA when it is transcribed?

We e-mailed Anderson to find out the exact tRNA gene sequences because we had a hard time finding this information everywhere else. Most papers we found describing the 5-base codon tRNA suppressors simply referred to Anderson's paper [http://gcat.davidson.edu/GcatWiki/images/6/6f/AndersonSchultz2002ChemBiol.pdf]. Anderson replied near the end of the day with the gene sequence. According to his email, the only difference between different tRNA genes is the anticodon loop. Therefore, if we just substitute all the anticodon loops in the invariable part of the sequence, we have our different tRNA sequences. We plan on using the Lancilator to help us break these tRNA sequences into smaller oligonucleotides that can anneal at around the same temperature because it is not possible for us to order oligos that are greater than 70 nucleotides long.

== [[User:Roclemente|Roclemente]] 17:21, 4 June 2009 (EDT) ==

I started out the day looking at my time-sensitive group research qustion about reporter proteins. I started making a table listing the advantages and disadvantages of different reporter proteins. Then the group found out that there was a major roadblock to our project: a stop codon was located in our ATG-5mer oligo. The stop codon would have been part of the BioBrick scar. The group looked at several ways to work around this problem. The idea that I looked most into was replacing the restriction sites that were used in the scar through standard assembly. According to the judging criteria for iGEM, I found that a team could possibly alter the standard assembly method as long as they wrote a letter to the iGEM judges explaining our plan in a detailed manner. From there, I set off to understand the BioBrick scar more by making a document highlighting the exact positions of the rsetriction sites on the BioBrick parts and how they were used to put more than one part together. I then found several other pairs of restriction sites that complemented one another and could be used in place of the standard scar between Xba1 and Spe1. At the end of the day, the group got the chance to speak with Dr. Campbell and he suggested a hybrid idea which would combine Davidson's restriction site idea with Missouri's PCR idea. I'm not quite sure of what this idea is exactly yet, but we'll all clarify our vague idea of it tomorrow morning after a talk with Dr. Eckdahl.

== [[User:Roclemente|Roclemente]] 17:13, 5 June 2009 (EDT) ==

This morning we began exploring different options for bypassing the stop codon problem from yesterday. If we wanted to make sure we were using the BioBrick parts in our GCatalog, there was no way we were going to be getting rid of the Xba1 restriction site that contained the "TAG" which codes for a stop codon. Therefore, we proposed looking at restriction sites within the reporter genes instead, therefore addressing the already-placed BioBrick ends. Using the document me and Shamita created on "Restriction Site Mapping on Reporter Genes" (see first journal entry), we determined that the reporter gene with the restriction site closest to the beginning of the sequence was YFP. We proposed cutting the YFP gene that we already had in stock at this restriction site and inserting an oligo (which we would have ordered) with a sequence in the following order: "BioBrick prefix - ATG - 5mer - part of YFP gene to the left of the cleaved restriction site."

After clarifying our own idea with one another, we spoke with some Missouri kids to clarify the said "hybrid" idea which, according to Dr. Campbell, combined the ideas of both campuses. The hybrid idea was based upon our idea of cutting the reporter gene at a restriction site as well, except that our primer would start out with a primer for our reporter gene, and to the left of the primer would be a "tail" consisting of the ATG and 5mer and either an Xba1 or EcoR1 restriction site (We still have to decide this. Our decision will probably be based on the restriction site we end up using in our reporter gene and whether or not that is complementary to either the Xba1 or EcoR1 sites.). We would use the PCR technique and use our in-stock reporter gene strand as our template strand (it will be denatured into single strands) and we will add our oligo. We propose that the primer will synthesize the rest of the YFP strand as well as add on nucleotides to complete the oligo tail. In the end, we will have a double stranded DNA sequence consisting of either the Xba1 or EcoR1 site, the ATG, the 5mer, and the reporter gene. The beauty of this hybrid idea is that we have flexibility as far as the reporter gene we choose to use since all we really need for it is the beginning sequence (for the primer) and a known, "hearty" restriction site. Once we cut this double strand at this restriction site on the reporter gene as well as at the Xba1 or EcoR1 sites, we can insert this into a plasmid that contains the rest of that reporter gene after the restriction site we choose to use and the complementary Xba1 or EcoR1 site.

I made a Word document outlining the promoter and RBS parts we have in our registry on the Davidson campus. Leland and I went through the registry parts on the GCATalog to look for potential promoters and RBS parts. We went through the document as a group and analyzed each of these potential parts on partsregistry.org to see which ones would most likely work the best in the miniprep we'll start preparing for on Sunday evening. Here is the document we wound up with:

http://gcat.davidson.edu/GcatWiki/images/d/d2/Promoters_and_RBS.doc

== [[User:Roclemente|Roclemente]] 10:40, 8 June 2009 (EDT) ==

We're starting the day by finalizing the oligos we will be ordering to have synthesized. Two groups of oligos must be sequenced: primers and tRNA gene sequence. The primers will consist of a forward and reverse primer. The forward primer will consist of the following in this 5' - 3' order: GCAT - BioBrick Prefix - ATF - 5mer - first 20 nucleotides in the reporter gene sequence. These 20 nucleotides won't include the ATG that naturally comes at the beginning of the reporter gene because we want to make sure the ribosome attaches to the first ATG and reads the logical clause instead of going straight to the ATG at the start of the reporter gene. The reverse primer will consist of the first 20 nucleotides downstream of the restriction site we plan to use in the reporter gene. I worked on finding the primers for the Tet resistance gene (part J31007) while Lyn worked on finding the primers for the RGP gene. Dr. Campbell decided we should use the BamH1 restriction enzyme because it is very "hardy." The following document outlines the 5 forward primers (each with one of the five variable 5mers) we will order for amplification of the Tet resistance FML:

[[Image:Tet resistance forward primers.png]]

This is the reverse primer we will order for the Tet resistance FML:

[[Image:Tet resistsance reverse primer.png]]

== [[User:Roclemente|Roclemente]] 11:54, 9 June 2009 (EDT) ==

We need to digest the plasmids at the EcoR1 and Pst siets. Everytime we're going to set up a digestion, we'll want the following in these volumes: 10x Buffer (2 uL), DNA (~ 3 uL), enzymes 1 and 2 (1 uL each), water (13 uL), and final volume of all of these put together should be 20 uL.

Some notes about digestion: Enzymes should make up 10% of the final volume. As a general rule of thumb, make sure the tip of your pipet is close to the surface of the solution. This is especially pertinent when gathering 1 microL of each of the enzymes. The most sensitive components are the enzymes, so they should go last.

We'll have a bunch of tubes with each 3 microL of the specific DNA. We'll make a cocktail in a bigger tube consisting of the buffer, 2 enzymes, and water. The rationale behind thissis that you'll be less likely to make a bigger mistake with a bigger volume (versus getting 1 microL of enzyme per specific DNA). You'll also know that there is no problem with the enzyme if all the DNA don't cut. If only one specific DNA doesn't work, then we'll know that it's because there's something wrong with the specific DNA rather than anything in the "Master Mix." Since we have to do 11 digestions, we'll make enough of the master mix for 12 tubes to make sure we have enough. We'll take 17 microL of the Master Mix to each tube of DNA.

The optimal temperature for most enzymes is 37 degreees celsius. You'd like to have enzymes with the same optimal temperatures (for ex. restriction enzymes that start with B usually have an optimal temperature near 50 degrees. You wouldn't want to use both B-enzyme and EcoR1 because EcoR1 would die).

'''What percent gel should we use?'''

Look at the Davidson lab protocol on the Davidson-Missouri Wiki. Generally, bigger molecules require an agarose gel with a lower concentration.

'''What's next in the future?'''

5' end additions are done by PCR. We'll ampliffy this double-stranded DNA. We'll cut with both EcoR1 and BamH1 or Nco1 (depending on which reporter gene you're using). Then we'll cut the plasmid with the reporter with the same restriction enzymes used.Then, to throw away the remaining DNA that we don't want, we'll run a gel and throw away the smaller band (because the larger band is probably made of the plasmid we want to keep).Then we'll take the double stranded DNA we amplified and the purified plasmid and ligate, transform, and screen them and verify the sequence.

While we wait for the oligos, we can cut the plasmid with reporter on the same restriction enzyme already.

'''How are we building the tRNAs?'''

We'll assemble the oligos into genes by mixing our oligos together, boiling them, and very slowly allowing them to cool. This will require many calculations. We'll cut the plasmids with EcoR1 and Pst1. to be ready to receive the enes we're assembling.We'll have to gel purify the plasmids to get them away from their inserts. We'll ligate, transform, and screen the genes and the plasmids and verify the sequence. On paper you shouldn't have two sites cut with different restriction enzymes stick together, but if there's selection pressure, these sites may cut some ends to fit together. This is why we must screen them.

After Dr. Campbell taught us all of the above, the 5 biologists put together a document outlining exactly how we would go about cutting the two sets of plasmids (one set will be cut with Eco R1 and Pst1 and othe other set will be cut with Eco R1 and BamH1 or Nco1). The following table outlines which biologist was going to work with which sample of DNA:

[[Image:Names and DNA.jpg]]

The following summarize the diferent materials and volumes of these materials that we'll need for our digestion of the plasmids:

[[Image:Digestion materials.png]]

This last table highlights the agarose percent concentration we should use for each part according to how many base pairs it contains:

[[Image:Screen-capture-3.png]]

The biologists digested the plasmids that we plan on using once our oligos arrive in a day or two. Afterwards, we made the two agarose gels that were most compatible with the parts we were going to run. This gel is going to purify the plasmids so that we have the parts that we're going to use later on, minus the rest of the DNA in the plasmid that we don't need. Before physically creating the gel, we figured out how much agarose we would need to make our two different agarose gel concentrations, 0.8% and 2.5%. Next, we made sure the mold and the big teeth were in place. We used the big teeth at the end of the mold because we plan on running 20 uL of DNA versus a running a smaller portion which would require us to use small teeth. We then measured the amount of agarose that we calculated we would need, placed this in a volumetric flask greater than 60 uL to allow for fizz to rise up, then measured out 60 uL of buffer and mixed this in the same flask with the agarose. We placed clear plastic wrap on top of the flask and heated it up for 1 minute and 20 seconds in the microwave. After we heated it, we took it out and checked to see if there was still agarose present that hadn't yet melted. There was, so we placed it back in the microwave and heated it for another 20 seconds. All the agarose had melted. We added 1 uL of EtBr (ethidium bromide) to the solution and quickly poured this into the mold. It needs about 15 to 20 minutes to cool. Dr. Campbell made the agarose gel that was 2.5% concentrated gel while we watched. We then made the 0.8% concentrated gel while he watched.

The following table lists the order in which we loaded our DNA into the gel. The first column all the way to the left is represented by number 1:

[[Image:Screen-capture-2.png]]

== [[User:Roclemente|Roclemente]] 08:46, 10 June 2009 (EDT) ==

After about 30 minutes after we began the gel electrophoresis, we looked at our gels with UV light to see how far the inserts had travelled and where the plasmids were. Then we took each gel to be photographed in the freezer room (231). We used the following DNA length marker to measure how long each sequence represented by each band was:

[[Image:Screen-capture-1.png]]

We compared our data to the lengths we expected to get according to the registry online. The first gel we photographed was the one that was 2.5% concentrated:

[[Image:2009-06-09 14hr 44min.jpg]]

The first two columns of the DNA we ran, both consisting of B0300, were fairly the same as the length we expected. However, the last two columns, consisting of K091111 and K091112 respectively were supposed to be 86 bp long, however, according to our data, K091112 was longer. Leland and I looked at Pallavi Penumetcha's paper which delved more closely into these two parts and what she wrote in her paper and what she entered into the registry conflict. We are currently corresponding with her to figure out the exact discrepancy.

We then photographed the 0.8% gel:

[[Image:2009-06-09 15hr 21min.jpg]]

The length of the sequences in almost every column matched up with the lengths we expected. Only the 4th and 5th columns didn't come out clearly. We think that the DNA inserted in column 5 weren't inserted properly and diffused on its own. We couldn't see bands in the 4th column at all until we adjusted the color a bit on the photographer and saw that column 4 was pretty much identical to column 5. We assumed that column 5 bled into column 4.

== [[User:Roclemente|Roclemente]] 15:14, 11 June 2009 (EDT) ==

We are digesting two parts today: I715039 (pLac+RBS+RFP) and S03710 (Tet Resistance). We want both the plasmids and the inserts in each case. We are aiming for a final volume of 40 uL for each part. This means that each digestion will consist of 4 uL buffer, 2 uL EcoR1, and 2 uL of their other respective restriction site (BamH1 for S03710 and Nco1 for I715039). The only parts that will vary are the amount of DNA and water we use. The amount of DNA we use depends on the concentration of the DNA we have (found through use of the NanoDrop). Since we want 2500 ng of DNA, we can use the concentration of DNA to figure out how much volume of this concentrated DNA we'll want to use in your digestion. If we subtract the volumes of buffer, two restriction enzymes, and DNA we plan on using from 40 uL, we'll have the amount of water we need to add. Here are the different volumes of materials we plan on using in our double digestion:

[[Image:DoubledigestRFPTetR.png]]

Now that we know how much of each material we were going to use, we needed to find out which buffer would be compatible with each sample according to the restriction enzymes we were using and how effective they'd be in different salt solutions. Online on the Promega "Compatible Buffers" website, we were able to find which Promega buffers would be most compatible when doing a double digest with EcoR1 and BamH1:

[[Image:BamEcoR1.png]]

Here is a chart noting buffers and their compatiblity with EcoR1 and Nco1:

[[Image:NcoEcoR1.png]]

We still have to wait for the restriction enzymes to arrive, so afterwards, we transformed three different inserts containing needed promoters and RBSs into vectors. We used the Zippy Transformation method. These promoters and RBSs will later be added to the FMLs once they are already inserted into the reporter cells. The following table outlines these inserts and part numbers:

[[Image:Promoterinsertspartnumbers.png]]

The restriction enzymes have arrived. We will now be able to do PCR to amplify the RFP gene (I715039) and the Tet resistance gene (S03710) as templates. We will be using the primers which have also arrived. We ordered forward and reverse primers so that our amplified sequence will consist of the following in this order:

GCAT-ATG-5mer-Reporter Gene

The following table lists the materials will be needed for this PCR technique which uses the "Green Promega master (Monster) Mix:"

[[Image:Greenpromegamastermix.png]]

This is the PCR procedure:

[[Image:Temperaturecycle.png]]

These are the variables that we will be adding to each separate sequence we want to be amplified:

[[Image:Tobeamplified.png]]

This table shows how we labeled the tubes based on how we divided the PCR work for the 10 tubes:

[[Image:Pcrtubes.png]]

Now we are giong to assemble the tRNA oligos together to form the tRNA genes. We could only order a certain length of sequence and the tRNA genes outdid these specified lengths. We used the Lancilator to find various oligonucleotides that overlapped and would be able to anneal together to form the tRNA genes. We used the Davidson lab protocol entitled "Buiding dsDNA with Oligos" to assemble the oligos:

[[Image:Oligoassembly.png]]

== [[User:Roclemente|Roclemente]] 22:46, 12 June 2009 (EDT) ==

We had two main goals for today:
1. perform a tRNA ligation and transformation
2. perform two electrophoreses: one to verify the PCR sequences we obtained and another to purify the digested reporter plasmids we would need

We began the day by pouring the agarose gels according to which concentration of buffer would be most compatible with the molecular weight of the substance we wanted to isolate. For both the digestion of E01010 and J31007, according to the "Optimize your Gel" website found on the Davidson Lab Protocols site for iGEM 2009, we wanted a 0.4% concentrated gel. For the PCRs, we wanted a 1.6% concentrated gel to optimize our retrieval of the Tet gene and a 1.4% concentrated gel to optimize our retrieval of the RFP gene.

While we waited for the gels to harden for about 30 minutes, we began calculating how much of each material we would need to ligate our tRNAs. We used the following equation to find out how much insert we should use:

[[Image:Ligationequation.png]]

Using the lengths of sequences we would be ligating, this is the mass that we needed:

X nanograms of insert = [(2)(144bp)(50 ng linearalized plasmid)]/2038bp
X = 7.0657 ng of insert

The following table outlines our calculations for finding the mass of tRNAs we had:

[[Image:Ligationcalculations.png]]

We would attain this mass of tRNA by pipetting an unknown amount of it. In order to attain a more "pipetable" amount of tRNA, we diluted our initial concentration 50X. Since we knew we wanted our final mass to be 7.065 ng, we calculated the volume we would need to take from this new, diluted sample:

DILUTION: 555.48/50 = 11.012
CALCULATION: 11.012 ng/ 1uL = 7.065 ng/ Y uL
Y = .64 uL of insert

Afterwards, Olivia and Alyndria began the actual ligation process while Shamita, Leland, and I filled the wells of the two gels. The first included the two digested reporter parts. We also included a molecular weight marker. Here is a photograph of this gel:

[[Image:2009-06-12 15hr 54min.jpg]]

According to our prior calculations, the Tet plasmid should have been 2958 base pairs long and the RFP plasmid should have been 2380 base pairs long. Our expectations were verified by the data. Shamita and I then sliced the part of thsi gel containing both plasmids and put these slices away to purfiy on Monday.

The second gel contained aliquots of the PCR we had done to attain 10 FML sequences: 5 containing the Tet resistant reporter gene and each of the 5 frameshifts that Davidson was assigned and the other 5 containing the RFP reporter gene and each of the 5 frameshifts as well:

[[Image:2009-06-12 16hr 07min.jpg]]

According to our data, each sequence containing the Tet resistant gene should have been 317 base pairs long and each sequence containing the RFP gene should have been 446 base pairs long. Our data supports our hypothesis, however, the 3rd well was not successful. This well contained the FML consisting of the Tet resistant gene and the frameshift mutant CUAGU. Before leaving work for the day, Shamita and I prepared and performed PCR using two parts containing the tet resistant gene, J31007 and S03710. We wanted to use both to make sure at least one of them works since we could technically use either of them, even though when we first did PCR we only used the S03710 part. Using the known concentrations of each of these parts, and knowing that we needed 1 ng of each part to include in our PCR samples, we calculated the volume of each 100X-diluted solution we would need:

S03710: 1.636 ng/uL = 1 ng/ X uL
X = 0.611 uL

J30017: 0.776 ng/uL = 1 ng/ Y uL
Y = 1.289 uL

We will run another gel with both of these parts on Monday and hopefully at least one of them works.

== [[User:Roclemente|Roclemente]] 17:24, 15 June 2009 (EDT) ==

Each biology member of the Davidson iGEM team has been assigned a 5mer to carry on throughout the rest of their respective processes. I've been assigned the 5mer CUAGU. We verified the PCR of the CUAGU FML containing the RFP gene on Friday. However, we weren't able to verify the CUAGU FML containing the tet resistance gene when we ran it on a gel. Shamita and I redid PCR for two parts containing the tet resistance gene: J31007 and S03710, labeled 2* and 2! respectively. I'm running another gel to verify either or both of them. Both sequences should be 317 bp long (this includes the ATF, logical clause, and the tet resistance gene to the left of the BamH1 restriction site). Here is the gel run we collected. The first column represents the 1 kb DNA marker, and the next two are 2* then 2!:

[[Image:2009-06-15 10hr 34min.jpg]]

The data shows that the PCRs of both parts wound up at the length we expected. The first time we did PCR on J31007, we must have left a solution out of our procedure on accident. I combined both PCRs together since they are the same length and match the length we expected to get.

Next, we cleaned and concentrated our amplified DNA sequence (#D4014 from Zymo Research). I cleaned the two pieces of DNA that contained the 5mer CUAGU. I labeled the tube with DNA also containing the RFP gene as "1" and the tube with DNA also containing the tet resistance gene as "2!". We needed 2 volumes of buffer for every 1 volume of DNA we added. Therefore, for tube "1," I added 95 uL DNA and 190 uL buffer. I added more DNA to tube "2!" since I combined the DNA from two PCR events to one (2* and 2! from earlier).

After cleaning the DNA, we found its concentration using the Nanodrop. In a solution made up of 0.5 uL of the DNA and 2.5 uL of TE, we found the following data from the DNA in this solution:

[[Image:Cleanconcentrate.png]]

The concentration of the DNA we cleaned was the concentration given by the Nanodrop multiplied by 6 (the DNA was dilute 6-fold for the Nanodrop). We used this concentration and the volume of clean DNA we knew we had (9.5 uL) to find the mass of clean DNA we had:

[[Image:Concentrateddna.png]]

Before doing digestion, we used the ligation equation (see "Ligation Protocol" in "Davidson Lab Protocols") to figure out how much mass of the DNA insert we would need later on when we did ligation. For the RFP FML, we found we would want the following mass of insert:

ng of insert = (2)(443)(50)/(2320) = 19.0948 ng

We multiplied this mass by 50 because we're counting on losing DNA between the digestion and cleaning we would need to do leading up to the ligation protocol.

19.0948 ng x 50 = 954.741 ng RFP FML

We found the mass of Tet FML insert we would need for ligation as well:

ng of insert = (2)(314)(50)/(2958) = 10.6153 ng

We multiplied this mass by 50 too:

10.6153 x 50 = 530.764 ng Tet FML

Unfortunately, nobody had enough of their clean DNA to satisfy our careful criteria. However, we would need to do this calculation later on so it was helpful we did it anyway. Instead, we just used all the DNA we had, or 9.5 uL for each reporter gene. Since there were 4 people who needed the same master mix, we made 5 parts of the master mix using the following volumes:

[[Image:Rfptetdigestion.png]]

After realizing that we would have to wait a day for the digestion to take place, we decided to culture every tRNA cell that colonized. The tRNA that we transformed on Friday did not have a good yield, so last night a few people came in and transformed more tRNA cells. We got a few more colonies. To be safe and make sure we cultured each tRNA, we picked every colony visible to us in both the old and new plates. Using our own given tRNAs, I counted the amount of colonies that grew in the plate we transformed on 6/12 and 6/14. There were 4 colonies that grew on both plates so I labeled a tube for each of these. I awas also in charge of the negative control. There were 2 colonies that grew on the negative control plate so I labeled 2 tubes for that. We will be placing them in an incubator overnight.

== [[User:Roclemente|Roclemente]] 12:30, 16 June 2009 (EDT) ==

We checked on the cells we cultured overnight. Of the cells that I cultured (all containing the CUAGU 5mer and the two negative controls), both negative control tubes worked as well as the tubes I labeled 1 (6/14), 5 (6/12), and 7 (6/12). The rest of mine were either red, meaning the plasmids hadn't ligated properly) or the colonies we used were no good. I took these 5 tubes, along with 3 of Olivia's tubes filled with cells that were cultured (tubes 11, 12, and 13) and miniprepped them to isolate the plasmids from the cells. There was no need to colony PCR to screen for successful ligations because this is only used to know which ones to let grow overnight and we had already done this.

As soon as I finished miniprepping them, I had to leave the lab for a little bit, so Olivia took each my miniprep tubes besides the negative controls and Leland took my negative controls. I completed the protocol for digestion of the tRNAs with EcoR1 and Pst1 for Alyndria's tubes when I got back because she had left a little earlier for lunch.

Afterwards, Shamita and I poured a 1.9% concentrated gel to load the digested tRNAs on. We want to verify that these tRNAs are the appropriate size for if they were cut properly. Since we will have to load 37 wells, and the maximum number we can load on one gel is 18 since 2 wells will have to be used by the molecular weight marker, we needed to pour 2 gels and we will also be using a half of a 2.0% agarose gel that was left over from another experiment.

Shamita and I also made new 0.5X running buffer gel. We did this by adding 450 uL buffer and 9 uL ethidium bromide (1 uL EtBr : 50 uL buffer). Alyndria, Leland, and Shamita loaded these gels.

Olivia and I cleaned our PCR DNA using the "Ethanol precipitate DNA (short protocol)" found in the Davidson Lab Protocols link. To begin with, I added 180 uL dH2O to my two samples, CUAGU with RFP and CUAGU with Tet, to bring their volumes up to 200 uL. During one of the last steps of this protocol, when Olivia and I dried out our DNA using the SpeedVac for a period of 10 minutes, the top of my tube containing the CUAGU 5mer and RFP gene broke. I wasn't sure if a pellet was in there, but I still poured out the ethanol and went on with the protocol. At the end, I resuspended what may or may not have been my DNA in 10 uL distilled water. I NanoDropped it afterward and attained the following data concerning both tubes:

[[Image:Failedpcrmeh.png]]

According to this data, any absorption and concentration we may have attained is insignificant because the A-260 must be between 0.05 and 1.50 to be significant and neither of the DNA is. Tomorrow I will do PCR for both FMLs over again.

== [[User:Roclemente|Roclemente]] 14:51, 17 June 2009 (EDT) ==

I found out that my tRNA DNA that I labeled "1" and which contains CUAGU was verified from the gel run Olivia did yesterday (the gel run also included her tRNA DNA samples). At the start of the day I plated this tube of cultured cells again to grow overnight.

Since PCR will take about 3 1/2 hours, I attempted this procedure at the beginning of the day. I plan on making 5 PCR products for each reporter gene since I now know the environmental conditions are correct. I labeled the RFP tubes 1-5 and the Tet tubes 6-10. I first needed to figure out how much of the template DNA I would need in order to have 1 ng template DNA. I made the following calculations to find this out:

[[Image:1ngtemplatedna.png]]

I wanted to add the Monster Mix last, so I made the following table, adding everything else besides the Monster Mix for 5 of the same reporter gene to one tube:

[[Image:Pcrmix.png]]

Unfortunately, when I actually mixed the solution together, I forgot to dilute the template DNA. Therefore, we have 100X more template DNA than we would have had we diluted the template DNA. The PCR can still work, but we will have to constantly remind ourselves that there will be extra template DNA in our solution and that this may skew, however slightly, our results later on.

While I waited for the PCR product, I refocused on getting my tRNA DNA ready for sequencing. Using the NanoDrop, I found the concentration of my miniprepped CUAGU DNA that was verified to be 61 ng/uL. The pre-sequencing protocol calls for 60 ng DNA, so I extracted 1 uL from this tube (the extra DNA will be insignificant in the sequencing procedure). I pipetted this into another tube and used the SpeedVac to dry it out. This is how we will send our DNA to be sequenced.

The following is a summary of steps I put together to help me plan out what I will do after the PCR procedure is over (it will be done at 5 pm today):

1. Combine the five matching tubes into one tube.
2. Run each PCR product on a gel to verify that our expected sequence has been properly amplified.
3. Clean and concentrate plasmids so salt solution is optimal for digestion.
4. Double digest PCR products with EcoR1 and BamH1 or Nco1.
5. Clean and concentrate again to get rid of restriction enzymes.
6. Nanodrop inserts in order to do ligation calculations and verify that sequences are present.
7. Ligate FML inserts with purified plasmids.
8. Transform plasmid into cells.

Since I won't have time to run a gel and take a picture once the PCR procedure is complete (we get out at 5:30 and it will be finished at 5:00), I have begun to prepare the agarose gel for use tomorrow. The size of the band I expect to find for the Tet FML is 349 bp (including the GCAT, BioBrick prefix, ATG, 5mer, and reporter gene until about 20 nucleotides or so past the restriction site we will cut at) and 474 bp for the RFP FML. According to the "Optimize Your Gel" link on the Davidson Lab Protocols page, a 1.6% gel will optimize how well I see my Tet bands and a 1.3% gel will optimize how well I see my RFP bands. I can use a gel with a concentration in between both of the optimal concentrations for Tet and RFP so that I can use one gel for both. Therefore, I will use a 1.45% gel. 1.45% of 60 uL buffer solution tells me that I should measure out 0.87 g agar for this gel.

== [[User:Roclemente|Roclemente]] 14:00, 18 June 2009 (EDT) ==

I checked on my PCR products this morning. Tubes 1 and 2 (both containing the RFP FML) had solution scattered on the tube's cover and walls so I placed them in the centrifuge for a bit. Unfortunately, I forgot to put these 500 uL tubes in holders when I centrifuged them so these tubes were destroyed. This was okay though because I was just going to combine each respective FML into one tube. I would have 300 uL of RFP FML PCR product instead of 500 uL and I put all of this in tube 5. I still had 500 uL of Tet FML PCR product left and put all of this in tube 10.

I ran 5 uL of both RFP and Tet FMLs on a gel, as well as a 1 kb molecular weight marker. The MW marker is in lane 1, the RFP is in lane 3, and the Tet is in lane 4 (Leland ran his PCR product in lane 2):

[[Image:2009-06-18 09hr 56min.jpg]]

I was expecting the RFP PCR product to be 474 bp long and the Tet PCR product to be 349 bp long. Both appear to come out a bit shorter, but Dr. Campbell and I agreed that we wouldn't count these sequences out because the bands were quite close to what we expected.

I proceeded to clean and concentrate these two sequences using the procedure outlined in the "Clean and Concentrate DNA (after PCR, before digestion)." Now I had 295 uL of the Tet sequence and 495 uL of the RFP sequence. Since the procedure says I can only have 800 uL in the column at a time, with DNA:Buffer at a ratio of 1:2, I mixed the two in a column in a series of two rounds using the following volumes:

[[Image:Clean.png]]

I Nanodropped my DNA after cleaning it and attained the following results:

[[Image:Nanome.png]]

I wanted the OD260 (Optical Density at 260) to be under 1.5, which it is for both. I lost quite a bit of Tet DNA in the process, and will only continue to lose DNA. To account for this loss of DNA, Dr. Campbell suggested it would be a good idea to heat inactivate the restriction enzymes to get rid of them after digestion instead of cleaning and concentrating the DNA over again.

I double digested both RFP and Tet FML sequences. I used all 9.5 uL of my DNA. The following table outlines how much volume of each material I mixed together:

[[Image:2nddoubledigest.png]]

While I waited 3 hours for my digestion to take place, I prepared 6 tubes to make more liquid cultures of the RFP and Tet vectors to grow more receiving vectors. 3 tubes would be to make RFP cultures and the other 3 would be for the Tet cultures. For RFP, I used the part I715039 and for Tet, I used the part J31007.

Once the digestion took place, Dr. Campbell notified me that heat inactivation was capable for Nco1 but not BamH1. There were 3 options for me to take in order to clean the Tet FML, the protocols for all of which can be found on the Davidson Lab Protocols link:

a) [[http://www.bio.davidson.edu/courses/Molbio/Protocols/Clean_Concentrate.html]]

File:Gel chart June 9.png

2009-06-18T19:50:15Z

ShPunjabi:

IGEM Notebook

2009-06-18T19:49:50Z

ShPunjabi: /* Tuesday, June 9, 2009 */

==Wednesday, June 3, 2009==

Romina Clemente and I are trying to find suitable reporter proteins to use. Yesterday, Leland Taylor and Alyndria Thompson were working on ways to insert the gene sequences into the plasmid. Upon seeing how they wanted to manipulated the reporter gene to include the logical clauses, we came up with a few criteria for the reporter genes we would use. The following criteria for genes are listed in order of the broadest aspect to look at to the narrowest aspect:
a) Doesn't contain restriction sites for the 4 restriction enzymes (EcoR1, Xbal, Spel, Pst1) used to cleave the Biobrick part out of the plasmid.
b) Contains 6 cutter restriction sites.
c) These restriction enzymes aren't blunt (cleave straight down at one spot).
d) These restriction sites are close to thge 5' (beginning) end of the sequence.
e) These enzymes are easiest to work with and cheapest.
We are finding the part numbers of the reporter genes we want to use (antibiotic resistance, fluorescence, LacZ) through our own GCAT because we know these ones work. We are then locating these parts on the parts registry [1] website. We copied and pasted the gene sequences we obtained from the registry onto the ApE software [2]. From here, we were able to generate a genetic map of each gene that outlined each restriction site that fit our criteria. We put each genetic map, alongside the part number used, into a Word document.

http://gcat.davidson.edu/GcatWiki/images/0/0e/Restriction_Site_Mapping_on_Reporter_Genes.doc

Later on in the day, we decided that the best way to test for suppression would involve placing the 5mers at the beginning of the reporter gene instead of inserting them into the reporter gene. To do this, we decided that the gene along with its start codon need to be expressed after the 5mer. We will insert a BioBrick plasmid with a 5mer and start codon that will be incorporated before the gene:
RBS-6-8nt-ATG-5mer--ATG-gene

Leland Taylor assembled the oligos that we will need for the BioBrick pieces.

In order to test whether the reporter protein would be expressed regardless of the 5mer being suppressed or not, we would need to remove the suppressor tRNAs from the cell.

Meanwhile, I helped figure out the coding sequence that we will need for the suppressor tRNAs that will bind to the 5mers. We decided that we would be using the following suppressor tRNAs on the Davidson side: CUAGU, CCCUC, CGGUC, CCAUC, and CCACU. We searched through several papers, with little result and finally emailed Dr. Christopher Anderson of UC Berkeley to request the DNA sequence. He emailed us back with a generic DNA code that we could use to create the tRNA; however, for each suppressor tRNA we would use, we would need to change base pairs in the anticodon loop.

The length of the tRNA is 92 nt. The anticodon loop is comprised of 9 nt. Once we have BioBricked the DNA sequence with 4 restriction sites and supplemental nucleotides, the length of the entire gene is 144 nucleotides. This is made up of the 92 nt +22 nt on each side of the sequence (44) + 4 nt on each side of the restriction ends (8).

At the end of the day, we decided to use tetracycline resistance and RFP as our reporter proteins for this tester experiment.

==Thursday, June 4, 2009==

This morning, I began the day by learning how to use the PEARL prgram. Olivia Ho-Shing sat with me and walked me through the program script. And then, Leland Taylor and Shashank Suresh had a problem they wanted us to solve. Leland explained that we need to make sure there is a stop codon in the gene if we fail to suppress the 5mer. The stop codon needs to be before we find another 5mer. They also wanted us to run through both the RFP and Tet Resistance genes to make sure that they did not contain any of the anticodon sequences from our 5 frameshift suppressor tRNAs so as to throw the translation out of frame. They further wanted us to confirm that if we do have successful suppression, there are no stop codons in the middle of the RFP and Tet Resistance genes. PEARL did not find any matches for the suppressor tRNAs within genes. We found 3 matches for stop codons UAA. Two of these occured at the end of the Tet and one at the end of RFP, where they should normally be to end translation into a protein.

Following this, Olivia Ho-Shing and I tried to determine how we can add BioBrick ends to our single stranded DNA sequences that code for the suppressor tRNAs. Our ultimate goal is to find complements for these single strands so they can be put into plasmids. We found the standard prefixes and suffixes for sequences that do not contain ATG (becuase this sequence is a functional RNA and will not be translated) and we placed these before and after the altered 92bp sequence that Dr. Anderson provided us with. The prefixes and suffixes are necessary because they are the extra nucleotides that will be mimicking a "restriction enzyme cutting site". The single strand appeared as below after the Bio Brick prefixes and suffixes were added:

22ntPREFIX--32nt--VARIABLE ANTICODON LOOP--51nt--21ntSUFFIX

We used the suppressor codon CUAGU as our example and placed its appropriate anticodon loop in the sequence. We put this into the lancelator that would give us the other strand as well as the oligos we would need for ideal construction of this double stranded DNA fragment to put into the plasmid. We recieved a total of 4 oligos, 2 of which were variable and 2 of which were constant for all 6 different suppressor tRNAs. Using this double strand, we simulated a restriction enzyme digestion by EcoRI and PstI. We took off the G at both ends of the top strand and then removed the first five nucleotides on each end of the bottom strand. This made the top strand longer than the bottom one. We then proceeded to take the two variable strands and changed the anticodon loop nt for each suppressor tRNA we used.

Around lunchtime, we encountered a problem with the BioBrick scar in the "ATG-5mer subpiece" of the test project. It turns out that after annealing the sticky ends of the Xba1 and Spe1 sites, the scar created had a stop codon TAG (UAG) that was in frame with the suppressed codon. When the Xba1 site on insert anneals to the Spe1 site on the plasmid (the green portions), we get a scar that reads TACTAG.
[[Image:screen-capture.png]]

For the afternoon, Olivia Ho-Shing and I finished assembly of the oligos of the suppressor tRNA codes. We assembled the document below:

http://gcat.davidson.edu/GcatWiki/images/5/5e/TRNAoligostoOrder.doc

We were assigned tasks of creating "cartoons" to illustrate the phenomenon that had occurred. No one could successfully do this however, because we could not understand how the piece had formed. The parts registry website provided wrong information on the restriction site of Spe1 which slowed our efficiency. This project turned into an effort to try to find two other different restriction enzymes that have complementary sticky ends other than Xba1 and Spe1. We want to get rid oc the scar that contains TACTAG.

We ended up deciding that a hybrid of two ideas was needed: restriction enzymes would need to be used to make several reporter gene sequences with different beginnings dependending on the suppressor tRNA sequence needed.

==Friday, June 5, 2009==

Our goal for this morning was to discuss the hybrid PCR approach to assemble the part that comes before the gene we wanted to express. We decided that we would have a BioBrick prefix and a 5mer followed by a sequence of nucleotides whose identity was up to us. Using PCR we would get the second double strand if we have the following:

BioBrick Prefix--ATG--5mer--gene until we get to restriction site

The PCR primer would attach to the ATG and the 5mer and would replicate the complementary base pairs to make a double stranded DNA sequence. As several of us were confused about PCR methods, we looked at the online link provided by Alyndria Thompson's link to the PCR interactive website which was very helpful. We also did some research to discover that the restriction enzyme BseRI (which is contained in the YFP that we wanted to alter) is not compatible with any other restriction enzyme. This is good because we did not want to plasmid containing this gene to close up with Xba1 once we digested it.

The team experienced some confusion with the hybrid idea and spoke to Clif Davis of Missouri Western University on the phone. He told us that there will be one part that is a fragment of the beginning of some nucleotides as a restriction cleaving site, ATG, the logical clause, and the start of the YFP gene that will be placed into a plasmid containing a space. To the left of the space will be the BioBrick Prefix and the right of the space will contain the rest of the genes in the YFP. Therefore, we decided not to alter any BioBrick ends and just look at restriction sites at the beginning of reporter proteins. The small fragment that will be placed into the plasmid will be made by PCR. The PCR will attach a primer onto the ATG and 5mer and replicate.

While we were discussing this, we expanded upon the idea that we can do whatever we want with the "suffix" portion between the 5mer and the beginning of YFP. We decided it would be best to remove ATG and add the first 5 amino acids and the restriction enzyme site. Along with this, if we have the 5mer coding for a certain amino acid (ie methionine) we could replace another codon that codes for methionine WITH that 5mer so long as this codon is contained within the first 15 nt. This way, we would not be altering the protein.

Olivia Ho-Shing came up with an idea of cascading PCR effect for longer logical clauses. Post translational modification of the protein was another one of her ideas. This applies because although we may code for the protein in a given way, the protein may modify or fix itself up to perform a slightly different function in its tertiary form.

Dr. Campbell suggested that we can look up restriction enzymes 400-500 base pairs into the reporter protein we would like to use; however, we have more flexibility in our procedure if we choose more "hardy" cutters. So instead, we could cut with EcoRI instead of with Xba1.

During the afternoon, we set about three tasks:
1) Which primers to use
2) Which promoters to use
3) Which restriction enzymes and how they will determine use of reporter proteins

Olivia Ho-Shing and I began looking up which restriction enzymes were strong so we could base our reporters on that. We began with Tet and RFP since those were the two reporters we had started out with choosing. I found a website that provided enzyme cleavage efficiency based upon the proximity of the site to the end of the PCR fragment that contains it. We discovered the following based upon the attached documents:

http://gcat.davidson.edu/GcatWiki/images/8/8f/Restriction_Enzyme_Cleavage_Efficiency.doc
http://gcat.davidson.edu/GcatWiki/images/9/9a/07Jun09_RE_Cutting_Efficiency_oh.doc

We decided that these two reporters contained efficient enzymes in a good quantity to choose from. On Monday, we will look through these enzymes once more and finalize which enzymes we would like to use with RFP and Tet.

==Sunday, June 7, 2009==

We did preparations for the mini prep this evening. The following notes are courtesy of Olivia Ho-Shing.

1. Sterically transfer ~2mL LB+AMP (AMP are ampicillin resistant plasmids) *Never put fluid back into bottle once it has entered pipette*
2. Use forceps to grab toothpick, scrap some frozen cells, rub in tube with top

'''Tubes'''

S03511 (2)

K091111

B0030

I715039

K091112

I715039

J31007 (2)

B0034

E1010

S03710

==Monday, June 8, 2009==

We reviewed the PCR process this morning as well as the restriction enzyme cleavage process.

Then, Leland Taylor, Olivia Ho-Shing and I all worked on converting the tRNA oligos we made to the template that Missouri Western used. The oligos we had previously made were different from the ones that Dr. Anderson had originally emailed us because further communication between Dr. Anderson and Missouri Western resulted in a better template.

We tried several times to minimize the number of oligos while keeping their individual lengths under 80bp. We managed to do this in the end by creating 7 oligos, 2 of which are variable between the suppressor tRNAs.
We reviewed the following document and finalized the oligos that we needed to order for the frameshift suppressor tRNAs. We decided to keep both the 10bp and 9bp anticodons of CCAUC.

I also looked up the amino acids that corresponded to the FS (frameshift suppressor) tRNAs for future use:

1. CUAGU = Leu
2. CCCUC = Pro
3. CGGUC = Arg
4. CCACU = Pro
5. CCAUC* = Pro
*9 and 10 bp anticodons

Also, we have decided to use NcoI as the restriction enzyme for RFP. This cleavage site occurs 419 bp into the gene. We have decided to use BamHI as the enzyme for Tetracycline Resistance and the cleavage site occurs 290bp into the gene. Both of these enzymes are very powerful and hardy.

We also looked over Missouri Western's tRNA oligos and primer oligos which we approved.
Our oligos were also approved by Missouri Western and Dr. Campbell ordered them while we supervised.

In the afternoon we did the miniprep. The mini prep directions were as appear in the link below.

http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_MiniPrep.html

We then worked with the Nano Prep to dilute our solution of DNA to an absorption of 1.5 and then we made labels for the tubes. The chart below shows relevant information we obtained from the NanoDrop for each of the reporter plasmids.

[[Image: Screen-capture-6.png]]

==Tuesday, June 9, 2009==

'''Preparations for restriction enzymes digestion of plasmids'''

Purpose: To determine the size of the plasmid inserts and ensure that plasmids are what we extracted from the cells during the mini-prep.

Things we need:
DNA ~3uL
Buffer 10x (Want to dilute to 1x) so add 2uL
Enzyme 1 ~1uL
Enzme 2 ~1uL
Water ~13uL
Final Volunter about 20 uL.

Need to choose the right buffer for the restriction enzymes. All buffers come in 10x (10 times more concentrated than what you want it to be when you use it). [http://gcat.davidson.edu/GcatWiki/index.php/Davidson_Missouri_W/Double_Digest_Guide Buffer Website]

Total volume of enzymes has to be 10% or less than final volume (most volume 2uL). Barely submerge the pipet tip into the solution when pipeting the enzymes.
Star (*) activity: Restriction enzyme stops cutting specifically and starts cutting everywhere.

DNA can't be seen by itself so we add EtBr (the bigger the insert, the more will bind) so bigger strands appear brighter. samples.

The order of entry doesn't matter, but make sure to enter the enzymes last.

Will have lots of labeled tubes and will put 3uL of appropriate plasmids into the tubes.
In the meantime, we'll make a "Master Mix" of all the other ingredients in a bigger tube. Always make enough volume for one extra tube.

Enzymes usually have an optimal temperature of 37C. We will let this reaction go for an hour and then we will run it on a gel.

'''Gel Electrophoresis Protocol'''

Website online for ideal gel concentration for resolving different sizes of molecules [http://gcat.davidson.edu/iGEM08/gelwebsite/gelwebsite.html Gel Concentration Website].
See protocol on the Davidson/Missouri Western Wookie for running molecules in the gel and how to digest with restriction enzymes.

'''What's Next?'''

1. 5' end additions
a. Using PCR
b. Cut at the restriction sites EcoR1 and either Bam H1 or Nco1
c. Cut plasmid and reporter with same restriction enzymes as above
d. Run a gel and purify "keeper" DNA (Ligate transform, screen, sequence)
e. Then, we take the "keeper" DNA and combine it with the insert from part

We can have steps d and e ready in advance.

2. tRNAs
a. Assemble the oligos into genes (Requires lots of calculations beforehand, but the actual steps )
b. Plasmids cut up with EcoR1 and Pst1 '''WE CAN USE THE "WASTE" PLASMID FROM THE RESTRICTION ENZYME DIGEST'''
c. Gel purify the plasmids (Ligate transform, screen, sequence)

The following tables include Master Mix measurements, agarose gel concentrations and buffers.
http://gcat.davidson.edu/GcatWiki/images/0/08/Shamitapic3.png
http://gcat.davidson.edu/GcatWiki/images/d/d1/Shamitapic4.png
http://gcat.davidson.edu/GcatWiki/images/6/6f/Shamitapic5.png

We made the Master Mix with our DNA and put it in the incubator at 10:40 AM.

'''Preparing the Gels'''

Now, we will be making 2 gels, 1 at 0.8% and 2.5% (weight to volume percent). The gel volume is 60mL.

'''Definition of 1% solution:
1 gram of agarose/ 100mL buffer'''

So for a 0.8% solution: 0.8 grams agarose/100mL buffer AND we need 60mL (fixed volume for casts run) so multiply that by 0.6.
Final concentration:

.48 grams agarose/60mL buffer

1 uL of EtBr stock for 60mL of gel.
EtBr is in the buffer so it is constantly replenishing itself even though it is moving toward one end in order to make bands on the other end visible.

After Dr. Campbell's demonstration, we each helped prepare a 0.8% buffer solution using the agarose powder, buffer and EtBr. We mixed together the first two substances in a 200mL Erlenmeyer flask and heated it for 120 seconds in the microwave. We then added EtBr and poured the gel solution into the gel cast.

'''Agar Plate'''

After lunch, we grew some more cells for each of the 11 parts in an agar plate labeled "LBAMP-AMC-9 June" so that so would have extra cells that could be stored for up to 2 weeks.
We made small circles on the agar plate and numbered them 1-11. The numbers corresponded to the following parts. We put the plate in the incubator overnight to let the cells grow.

1. B0030 AMC
2. K091111
3. S03710
4. I715039 OEH
5. S03511 ORH
6. I715039 AST
7. B0030 SDP
8. K091112
9. J31007 LJH
10. J31007 REC
11. S03511

'''Unloading the Gels'''

The following shows which parts we entered in which gels:

[[Image: Gel chart June_9.png]]

We unloaded the 2.5% agarose first and after observing the bands under a UV light, put the gel into the BioRad machine to observe the bands. The image below is of a Molecular Weight Marker that shows the number of bp in control fragments of DNA according to the distance they travel down the gel.

[[Image: DNA_molecular_weight_marker_gel.png]]

We obtained the following printout for the first gel that was 2.5% Agarose.
[[Image: 2009-06-09_14hr_44min.jpg]]

The bands in the first well at the bottom are those of the molecular weight marker. We skipped the lane above (to the right) of the MWM and entered the rest of the inserts as described in the above table. A light band is visible for each of the 4 other wells. In Lanes 3 and 4 we concluded we are supposed to have approximately 45 bp because the insert is 15bp + 30 bp on either side of the insert (Xba1 and Spe1 restrictions sites + sticky ends + extra bases). We used the same logic for Lanes 5 and 6 which are supposed to contain approximately 86 base pairs.

One discrepancy in this gel, however, is that Lanes 5 and 6, which according to their labels respectively contain the LacIQ promoter and the Lac IQ1 promoter. According to the information online at the parts registry website, pLacIQ1 should be the same length as pLacIQ. The gels do not show this, but instead show that the sample in Lane 5 is smaller than that in Lane 6 because Lane 5 traveled further. Leland Taylor and Romina Clemente sent out an email to Pallavi Penumetcha to ask about this because she had written a paper about this.

We then unloaded the 0.8% agarose gel and then placed this in the BioRad machine under the same protocol as above. The following image is the 0.8% Agarose.

[[Image:2009-06-09_15hr_21min.jpg]]

We observed that the bands in Lane 1 (which is now at the top) are examples of high intensity bands. The plasmids contained in the original well are twice the intensity of the insert.

In Lane 5, however, we observed several different bands. This could be due to the extra DNA that was sitting on the top of the well. We decided to re-expose the gel to get better observations on Lanes 3 and 4 because the bands were not as intense. We anticipated seeing a band at about 250 bp because this was the size of the insert (plus extra bp). After re-exposing the gel, we did not find any difference and still did not see any bands in these lanes. We have decided to redo the gel on the part S03511 and so we prepared the master mix with that DNA and incubated it so that we could run the gel tomorrow.

We also cut out the "empty" plasmids from the gel so that we could use this as vectors for the coding sequence of the suppressor tRNAs.

Tomorrow we will be planning out specific tasks for people once the oligos arrive.

==Wednesday, June 10, 2009==

We began this morning by making the agarose gel with the same protocol as yesterday. This time, since we are just re-doing both S03511 inserts, we will have a 1.8% agarose gel.

'''OUTLINE: WHERE DO WE STAND?'''

Yesterday
a. Isolated empty plasmids
b. Verified most inserts
c. Waiting for:
>tRNA oligos
>Primers
a. Will amplify the reporter gene in the plasmid itself

Next?
a. When Primers Arrive
> Amplify the reporter genes with primers (PCR takes ~3 hours)
> Calculate how much reporter plasmid we will need (Start with 50x more than we actually will need)
> Cut amplified sequence (EcoR1 and BamH1/Nco1) then purify what we need
> Cut reporter plasmid (EcoR1 and BamH1/Nco1) then purify what we need
> Ligate amplified sequence and reporter plasmid
b. When tRNA oligos arrive
> Calculate how much of each oligo we will need (takes a while...)
> "Boil and cool" tRNA oligos together
> Ligate fragments (dsDNA) into plasmids we isolated yesterday (~5 minutes)
> Transform the plasmids (~20 minutes)

* LOOK THROUGH LIGATION PROTOCOL (How to assemble genes)
* LOOK THROUGH TRANSFORMATION PROTOCOL

''What we need to get done today'':
1. Prepare the control for the Tet Resistance by BioBricking the part pLac+RBS (S03511) with the TetA (J31007).
2. Check the part number for RFP and also for pLac+RBS+RFP
3. Read through ligation protocol
4. See how many nanograms of plasmid we will need for the tRNAs and for the inserts and multiply this number by 50.
> Check if we have enough plasmids from our mini-prep
5. Purify the plasmids from the gel

After we made this outline, Leland, Olivia and I analyzed the gel we ran this morning and did not see a more than one band in the 2 lanes that contained DNA from S03511 (image below). Since we did not see multiple bands in yesterday's gel either, we conclude that there is something wrong with the DNA rather than with the restriction enzymes that we used to cut it. We concluded that there is probably not an insert in this plasmid both the part and the plasmid are useless.

[[Image: 2009-06-10 10hr 30min.jpg]]

Now, we do not have an immediately accessible promoter. We have decided to order the pBAD promoter from Missouri Western and we will attach the RBS+Tet to this plasmid. Our insert for this will be the larger gene because it is easier to manipulate in the gel than the small gene.

Alyndria Thompson and Romina Clemente are figuring out the size of the plasmids including the reporter genes.

After lunch we performed several steps to isolate and purify the gel to get the empty plasmids will be the vectors for the tRNA DNA. Then, we used the NanoDrop to assess the purity of our DNA. We found that the plasmids were more pure than we had expected. The following values were obtained:

Abs 1.850
A-260 10 mm path 0.172
A-280 10 mm path 0.075
260/280 2.28
260/230 0.09

We then followed the protocol for preparing the agar plates. We put the plate with the agar solution in the Autoclave.

While the solution was in the autoclave, we discussed that we don't really need a promoter in front of RBS+Tet for the gene to be expressed because Tet expression is so strong that the gene would translate without a promoter. However, it would be a good idea to have the promoter because if it was possible to reinforce the translation of the reporter protein, we wanted to do that.

We found that there were some tubes in the lab fridge that contained the pBAD and pLac promoters with RBS. We decided to use these as the promoter and RBS for our experiments. The pLac promoter will be used for RFP and the pBad promoter used with Tet Resistance. We decided to use the pBad with Tet because it is weaker in comparison to pLac. As expression of the Tet gene can be toxic to a cell, we do not want to overexpress the gene with a stronger promoter.

Once the agar solution was sterilized in the Autoclave, we removed it, cooled it, and pipetted the antibiotic resistance into it. We then poured the solution into the plates, removed any bubbles, and let the gel form.

Alyndria Thompson and Romina Clemente concluded that we had more than enough recieving vectors for both the RFP and Tetracycline genes. Since we need 50 nanograms, it is wise to have at least 50 times that much that we can work with, so we need at least 2,500 ng.

RFP 1: 77.6 x 4 = 310.4 ng/uL
x 20 uL = 3,272 ng
RFP 2: 59.5 x 4 = 238 ng/uL
x 20 uL = 4,760 ng
Tet: 40.9 x 4 = 163.6 ng/uL
x 20 uL = 3,272 ng

According to these calculations we have more than 50 times what we need for both instances of RFP and Tet.

Tomorrow, we can prepare the Master Mix for the digestions of both reporter plamids and then add the restriction enzymes once they arrive. Then, we can follow through with the rest of the plans we had made above in the section titles "Next?".

==Thursday, June 11, 2009==

We began this morning by calculating how many uL we would need for each of the mixes for digesting our reporter plasmids. We decided to double the final volume of each mix to 40uL instead of 20uL. Since we knew the concentration of each plasmid and also knew we needed 2500 ng, we cross multiplied to find the uL of DNA we would need for each.

Concentration Calculations:
310.4 ng/1 uL = 2500ng/x uL
x = 8.05 uL

Here is a chart indicating how much of each we used for the 2 mixes.

[[Image: DoubledigestRFPTetR.png]]

On the Promega website, we found a list of compatible buffers for double digestions. Buffers E and H are compatible for the Tet plasmid and RFP plasmid digestions respectively. We fortunately have both buffers in stock.

[[Image:BamEcoR1.png]] [[Image:NcoEcoR1.png]]

We waited for our waited for the restriction enzymes to arrive and in the meantime, we transformed the plasmids with pLac+RBS, pBad, and pBad+RBS into ''E.coli'' cells. We want the ''E.coli'' cells to replicate the plasmids with these genes because we have a limited amount of genes. [http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_Transformation.html Zippy Plasmid Transformation]

Once our oligos and restriction enzymes arrived, we got to work right away!

'''PCR (Polymerase Chain Reaction)'''

We calculated how much plasmid we will need for the PCR. We decided to dilute the concentration of our plasmids a hundred fold so that we could get pipetable amounts.

''Change in Concentrations''
310.4 ng/uL x 1/100 (dilution factor) = 3.104 ng/uL (RFP)
163.6 ng/uL x 1/100 (dilution factor) = 0.164 ng/uL (Tet)

''Example shown with RFP''
3.104 ng/1 uL = 1ng/ x uL, x = .32
For Tet, x = .61

We also calculated how much of each primer we would need with the following equation:

C1V1 = C2V2
(100 uM) (x) = (1 uM)( 100 uL)
x = 1 uL

[[Image: screen-capture-8.png]]

Each of the 5 of us made had 2 tubes (one for each reporter protein) because there were 5 different forward primers.To each tube, we added the template DNA (either RFP or Tet), water, the respective reverse primer (either RFP or Tet), and the forward primer depending on which 5mer we had chosen. My 5mer was CCAUC. Finally, we added the Master Mix last because this contained the enzyme (DNA polymerase) that would amplify our desired sequence.

[[Image: Pcrtubes.png]]

I had CCAUC as 5mer.

We followed the temperature cycles so that PCR could occur. The annealing temperature needed to be 5C less than the smallest melting temperature of the primers. Since the reverse primer of RFP had a melting temp of 66C, we set the temperature of the 3rd cycle on 61C.

[[Image: Temperaturecycle.png]]

'''tRNA Oligo Assembly'''

We needed to calculate the concentration of each of the oligo before carrying out the assembly. The stock concentration of each of the oligo was 100 uM. Since we wanted to have 5uM of the oligo in the test tube, and we know that final volume of the test tube is 20 uL, we just cross multiplied to find the amount we needed to add to the tubes to get this concentration.

(100uM)x = (5uM)(20uL)
x = 1 uL

We added 1 uL of each oligo to the tubes.

We had six total tRNAs we needed to assemble so each of us took one FML (one person took two) and to each tube we added 1uL of each oligo for a total of 7uL, 2uL of buffer, and 11uL of water for a total volume of 20 uL. Five of the oligos were constant and 2 varied for each of the different tRNAs. So everyone pipetted the same 5 oligos and everyone also had 2 unique oligos to add. When all of the oligos were added and the tubes ready, we boiled the tubes at 50C for 5 minutes. We then allowed the tubes to remain in the water to cool very slowly overnight so that the annealing process could take place.

The last thing we did at the end of the day was finish off our plasmid digestion that we had begun in the morning. We added the buffer, DNA, water, and restriction enzymes and placed this in the incubator for overnight digestion.

'''To Do''':

1. Isolate our desired dsDNA from the PCR.
2. Verify our tRNA inserts and eventually transform these into plasmids.
3. Check our agar plates to see colonies that have replicated our pLac+RBS, pBAD+RBS, and pBAD genes.

==Friday, June 12, 2009==

This morning, we began by outlining the main items on today's agenda.

1. tRNAs: Ligation, Transformation
2. Digestion reporters (run all of it and gel purify)
3. Transformed cells
4. PCR: Gel run an aliquot to verify (5uL)

We used the equation given by the Ligation protocol to calculate how many nanograms of insert we would need. Initially, we started out calculated the MW average of the 9 and 10 anticodon loops of tRNAs which were 15828.23 ug/umols and 159016.6 ug/umols respectively. We then saw, however, that these would not be helpful values in the process. Instead we started out with the equation below to find the final ng of insert we would need for the ligation.

[[Image: ligationcalculations.png]]

We calculated our initial concentration in ng/uL of insert as shown below.

[[Image: ligationequation.png]]
X nanograms of insert = [(2)(144bp)(50 ng linearalized plasmid)]/2038bp
X = 7.0657 ng of insert

We wanted to find the amount of uL of insert we would need to use. We diluted the initial concentration 50x because we wanted a pipettable amount of insert. Since we knew we wanted the final amount of nanograms to be 7.0657, we calculated as shown below:

''DILUTION'': 555.48/50 = 11.012
''CALCULATION'': 11.012 ng/ 1uL = 7.065 ng/ x uL
x = .64 uL of insert

After we had done these calculations, half of the group started to carryout the ligation of the tRNA inserts into empty plasmids. We needed 7 ligations total: 6 were the tRNAs we needed, while the last one is a negative control. These are the final amounts of what we used:

[[Image: Trnaligation.png]]

The other half of the group (which included me) prepared the agarose gels so that we could run gels for the digested reporters and the PCR inserts. We calculated that the digested reporters needed an agarose of 0.4% and the PCR reporters needed an agarose of 1.45%. We allowed this gel to form for about 45 minutes to an hour. We prepared both the gels and allowed them to run through lunch.
After lunch, we took pictures of our gels and noticed that the third lane in the PCR gel (third lane from the top in the bottom photo) did not develop a band. The lane was supposed to contain the CUAGU insert for Tetracycline Resistance.

[[Image: 2009-06-12 15hr 54min.jpg]] [[Image:2009-06-12_16hr_07min.jpg]]

Romina Clemente and I decided to repeat this PCR using the TetResistance and RBS+TetResistance plasmids as templates. We labeled J31007 as "2*" and S03710 as "2!". We had to recalculate how much template DNA to use. We let the PCR run overnight. We also verified that the digestion fragments (both the plasmid and the insert) on the other gel were the correct size. The plasmids, which are the brighter fragment in the gel with only 2 lanes, contain the plasmid base pairs along with the rest of our reporter genes. We isolated these plasmids from the gel today and stored them for purification on Monday.

In the meantime, other members of our lab had finished the ligation of the tRNA genes to the empty plasmids and were now transforming these into competent cells. They placed the agar plate with these cells in the fridge overnight.

We also observed the transformations that we had performed yesterday and noticed that I13453 did not form any colonies. J04500 and J3102 did form colonies.

To Do:
1. PCR: Dig, Ligate, Transform
Clean and concentration column, digest, clean and concentration column, ligate.
2. Gel purification of our digestion plasmids
3. Check if colonies have grown for our tRNAs

==Saturday, June 13, 2009==

Alyndria Thompson came in on Saturday morning to remove the tRNA plates and put the PCR reactions in the fridge.

'''tRNA Colonies'''

She counted the colonies of tRNA on each plate.

[[Image:TRNA_colonies_6_13_09.png]]

She placed the plates in the large fridge on the top shelf at 9:23 am. She talked to Dr. Campbell who said we will have to try to "work them up".

'''PCR'''

Products were placed in orange rack of smaller fridge. [2*, 2!]

==Sunday, June 14, 2009==

'''Second Ligation and Transformation of tRNA inserts'''

We decided to re-ligate and re-transform all of the tRNA inserts because we did not get very good yields of colonies from the transformations.
We are using the same measurements for this ligation as we had used on Friday.

[[Image: Trnaligation.png]]

After ligation, we transformed the cells. Using the following measurements, we transferred the competent cells into new tubes.

40uL of competent cells
10uL of ligation mixture
60uL of LB media

After spreading the mixture onto the agar plates, we incubated the plates at 10:56 pm.

We also stored liquid cultures of the colonies that greater than a 2:1 variable:negative control ratio just in case we wanted to use them for mini preps in the future. These included #1 (3 samples), #4 (2 samples), #5 (1 sample), and #6 (2 samples).

'''To Do''':
1. Count colonies obtained from second transformation
2. Clean PCRs: Dig, ligate, transform
3. Run gel for 2 Tet PCRs, clean
4. Gel purify receiving vector

IGEM Notebook

2009-06-15T21:54:19Z

ShPunjabi: /* Thursday, June 11, 2009 */

==Wednesday, June 3, 2009==

Romina Clemente and I are trying to find suitable reporter proteins to use. Yesterday, Leland Taylor and Alyndria Thompson were working on ways to insert the gene sequences into the plasmid. Upon seeing how they wanted to manipulated the reporter gene to include the logical clauses, we came up with a few criteria for the reporter genes we would use. The following criteria for genes are listed in order of the broadest aspect to look at to the narrowest aspect:
a) Doesn't contain restriction sites for the 4 restriction enzymes (EcoR1, Xbal, Spel, Pst1) used to cleave the Biobrick part out of the plasmid.
b) Contains 6 cutter restriction sites.
c) These restriction enzymes aren't blunt (cleave straight down at one spot).
d) These restriction sites are close to thge 5' (beginning) end of the sequence.
e) These enzymes are easiest to work with and cheapest.
We are finding the part numbers of the reporter genes we want to use (antibiotic resistance, fluorescence, LacZ) through our own GCAT because we know these ones work. We are then locating these parts on the parts registry [1] website. We copied and pasted the gene sequences we obtained from the registry onto the ApE software [2]. From here, we were able to generate a genetic map of each gene that outlined each restriction site that fit our criteria. We put each genetic map, alongside the part number used, into a Word document.

http://gcat.davidson.edu/GcatWiki/images/0/0e/Restriction_Site_Mapping_on_Reporter_Genes.doc

Later on in the day, we decided that the best way to test for suppression would involve placing the 5mers at the beginning of the reporter gene instead of inserting them into the reporter gene. To do this, we decided that the gene along with its start codon need to be expressed after the 5mer. We will insert a BioBrick plasmid with a 5mer and start codon that will be incorporated before the gene:
RBS-6-8nt-ATG-5mer--ATG-gene

Leland Taylor assembled the oligos that we will need for the BioBrick pieces.

In order to test whether the reporter protein would be expressed regardless of the 5mer being suppressed or not, we would need to remove the suppressor tRNAs from the cell.

Meanwhile, I helped figure out the coding sequence that we will need for the suppressor tRNAs that will bind to the 5mers. We decided that we would be using the following suppressor tRNAs on the Davidson side: CUAGU, CCCUC, CGGUC, CCAUC, and CCACU. We searched through several papers, with little result and finally emailed Dr. Christopher Anderson of UC Berkeley to request the DNA sequence. He emailed us back with a generic DNA code that we could use to create the tRNA; however, for each suppressor tRNA we would use, we would need to change base pairs in the anticodon loop.

The length of the tRNA is 92 nt. The anticodon loop is comprised of 9 nt. Once we have BioBricked the DNA sequence with 4 restriction sites and supplemental nucleotides, the length of the entire gene is 144 nucleotides. This is made up of the 92 nt +22 nt on each side of the sequence (44) + 4 nt on each side of the restriction ends (8).

At the end of the day, we decided to use tetracycline resistance and RFP as our reporter proteins for this tester experiment.

==Thursday, June 4, 2009==

This morning, I began the day by learning how to use the PEARL prgram. Olivia Ho-Shing sat with me and walked me through the program script. And then, Leland Taylor and Shashank Suresh had a problem they wanted us to solve. Leland explained that we need to make sure there is a stop codon in the gene if we fail to suppress the 5mer. The stop codon needs to be before we find another 5mer. They also wanted us to run through both the RFP and Tet Resistance genes to make sure that they did not contain any of the anticodon sequences from our 5 frameshift suppressor tRNAs so as to throw the translation out of frame. They further wanted us to confirm that if we do have successful suppression, there are no stop codons in the middle of the RFP and Tet Resistance genes. PEARL did not find any matches for the suppressor tRNAs within genes. We found 3 matches for stop codons UAA. Two of these occured at the end of the Tet and one at the end of RFP, where they should normally be to end translation into a protein.

Following this, Olivia Ho-Shing and I tried to determine how we can add BioBrick ends to our single stranded DNA sequences that code for the suppressor tRNAs. Our ultimate goal is to find complements for these single strands so they can be put into plasmids. We found the standard prefixes and suffixes for sequences that do not contain ATG (becuase this sequence is a functional RNA and will not be translated) and we placed these before and after the altered 92bp sequence that Dr. Anderson provided us with. The prefixes and suffixes are necessary because they are the extra nucleotides that will be mimicking a "restriction enzyme cutting site". The single strand appeared as below after the Bio Brick prefixes and suffixes were added:

22ntPREFIX--32nt--VARIABLE ANTICODON LOOP--51nt--21ntSUFFIX

We used the suppressor codon CUAGU as our example and placed its appropriate anticodon loop in the sequence. We put this into the lancelator that would give us the other strand as well as the oligos we would need for ideal construction of this double stranded DNA fragment to put into the plasmid. We recieved a total of 4 oligos, 2 of which were variable and 2 of which were constant for all 6 different suppressor tRNAs. Using this double strand, we simulated a restriction enzyme digestion by EcoRI and PstI. We took off the G at both ends of the top strand and then removed the first five nucleotides on each end of the bottom strand. This made the top strand longer than the bottom one. We then proceeded to take the two variable strands and changed the anticodon loop nt for each suppressor tRNA we used.

Around lunchtime, we encountered a problem with the BioBrick scar in the "ATG-5mer subpiece" of the test project. It turns out that after annealing the sticky ends of the Xba1 and Spe1 sites, the scar created had a stop codon TAG (UAG) that was in frame with the suppressed codon. When the Xba1 site on insert anneals to the Spe1 site on the plasmid (the green portions), we get a scar that reads TACTAG.
[[Image:screen-capture.png]]

For the afternoon, Olivia Ho-Shing and I finished assembly of the oligos of the suppressor tRNA codes. We assembled the document below:

http://gcat.davidson.edu/GcatWiki/images/5/5e/TRNAoligostoOrder.doc

We were assigned tasks of creating "cartoons" to illustrate the phenomenon that had occurred. No one could successfully do this however, because we could not understand how the piece had formed. The parts registry website provided wrong information on the restriction site of Spe1 which slowed our efficiency. This project turned into an effort to try to find two other different restriction enzymes that have complementary sticky ends other than Xba1 and Spe1. We want to get rid oc the scar that contains TACTAG.

We ended up deciding that a hybrid of two ideas was needed: restriction enzymes would need to be used to make several reporter gene sequences with different beginnings dependending on the suppressor tRNA sequence needed.

==Friday, June 5, 2009==

Our goal for this morning was to discuss the hybrid PCR approach to assemble the part that comes before the gene we wanted to express. We decided that we would have a BioBrick prefix and a 5mer followed by a sequence of nucleotides whose identity was up to us. Using PCR we would get the second double strand if we have the following:

BioBrick Prefix--ATG--5mer--gene until we get to restriction site

The PCR primer would attach to the ATG and the 5mer and would replicate the complementary base pairs to make a double stranded DNA sequence. As several of us were confused about PCR methods, we looked at the online link provided by Alyndria Thompson's link to the PCR interactive website which was very helpful. We also did some research to discover that the restriction enzyme BseRI (which is contained in the YFP that we wanted to alter) is not compatible with any other restriction enzyme. This is good because we did not want to plasmid containing this gene to close up with Xba1 once we digested it.

The team experienced some confusion with the hybrid idea and spoke to Clif Davis of Missouri Western University on the phone. He told us that there will be one part that is a fragment of the beginning of some nucleotides as a restriction cleaving site, ATG, the logical clause, and the start of the YFP gene that will be placed into a plasmid containing a space. To the left of the space will be the BioBrick Prefix and the right of the space will contain the rest of the genes in the YFP. Therefore, we decided not to alter any BioBrick ends and just look at restriction sites at the beginning of reporter proteins. The small fragment that will be placed into the plasmid will be made by PCR. The PCR will attach a primer onto the ATG and 5mer and replicate.

While we were discussing this, we expanded upon the idea that we can do whatever we want with the "suffix" portion between the 5mer and the beginning of YFP. We decided it would be best to remove ATG and add the first 5 amino acids and the restriction enzyme site. Along with this, if we have the 5mer coding for a certain amino acid (ie methionine) we could replace another codon that codes for methionine WITH that 5mer so long as this codon is contained within the first 15 nt. This way, we would not be altering the protein.

Olivia Ho-Shing came up with an idea of cascading PCR effect for longer logical clauses. Post translational modification of the protein was another one of her ideas. This applies because although we may code for the protein in a given way, the protein may modify or fix itself up to perform a slightly different function in its tertiary form.

Dr. Campbell suggested that we can look up restriction enzymes 400-500 base pairs into the reporter protein we would like to use; however, we have more flexibility in our procedure if we choose more "hardy" cutters. So instead, we could cut with EcoRI instead of with Xba1.

During the afternoon, we set about three tasks:
1) Which primers to use
2) Which promoters to use
3) Which restriction enzymes and how they will determine use of reporter proteins

Olivia Ho-Shing and I began looking up which restriction enzymes were strong so we could base our reporters on that. We began with Tet and RFP since those were the two reporters we had started out with choosing. I found a website that provided enzyme cleavage efficiency based upon the proximity of the site to the end of the PCR fragment that contains it. We discovered the following based upon the attached documents:

http://gcat.davidson.edu/GcatWiki/images/8/8f/Restriction_Enzyme_Cleavage_Efficiency.doc
http://gcat.davidson.edu/GcatWiki/images/9/9a/07Jun09_RE_Cutting_Efficiency_oh.doc

We decided that these two reporters contained efficient enzymes in a good quantity to choose from. On Monday, we will look through these enzymes once more and finalize which enzymes we would like to use with RFP and Tet.

==Sunday, June 7, 2009==

We did preparations for the mini prep this evening. The following notes are courtesy of Olivia Ho-Shing.

1. Sterically transfer ~2mL LB+AMP (AMP are ampicillin resistant plasmids) *Never put fluid back into bottle once it has entered pipette*
2. Use forceps to grab toothpick, scrap some frozen cells, rub in tube with top

'''Tubes'''

S03511 (2)

K091111

B0030

I715039

K091112

I715039

J31007 (2)

B0034

E1010

S03710

==Monday, June 8, 2009==

We reviewed the PCR process this morning as well as the restriction enzyme cleavage process.

Then, Leland Taylor, Olivia Ho-Shing and I all worked on converting the tRNA oligos we made to the template that Missouri Western used. The oligos we had previously made were different from the ones that Dr. Anderson had originally emailed us because further communication between Dr. Anderson and Missouri Western resulted in a better template.

We tried several times to minimize the number of oligos while keeping their individual lengths under 80bp. We managed to do this in the end by creating 7 oligos, 2 of which are variable between the suppressor tRNAs.
We reviewed the following document and finalized the oligos that we needed to order for the frameshift suppressor tRNAs. We decided to keep both the 10bp and 9bp anticodons of CCAUC.

I also looked up the amino acids that corresponded to the FS (frameshift suppressor) tRNAs for future use:

1. CUAGU = Leu
2. CCCUC = Pro
3. CGGUC = Arg
4. CCACU = Pro
5. CCAUC* = Pro
*9 and 10 bp anticodons

Also, we have decided to use NcoI as the restriction enzyme for RFP. This cleavage site occurs 419 bp into the gene. We have decided to use BamHI as the enzyme for Tetracycline Resistance and the cleavage site occurs 290bp into the gene. Both of these enzymes are very powerful and hardy.

We also looked over Missouri Western's tRNA oligos and primer oligos which we approved.
Our oligos were also approved by Missouri Western and Dr. Campbell ordered them while we supervised.

In the afternoon we did the miniprep. The mini prep directions were as appear in the link below.

http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_MiniPrep.html

We then worked with the Nano Prep to dilute our solution of DNA to an absorption of 1.5 and then we made labels for the tubes. The chart below shows relevant information we obtained from the NanoDrop for each of the reporter plasmids.

[[Image: Screen-capture-6.png]]

==Tuesday, June 9, 2009==

'''Preparations for restriction enzymes digestion of plasmids'''

Purpose: To determine the size of the plasmid inserts and ensure that plasmids are what we extracted from the cells during the mini-prep.

Things we need:
DNA ~3uL
Buffer 10x (Want to dilute to 1x) so add 2uL
Enzyme 1 ~1uL
Enzme 2 ~1uL
Water ~13uL
Final Volunter about 20 uL.

Need to choose the right buffer for the restriction enzymes. All buffers come in 10x (10 times more concentrated than what you want it to be when you use it). [http://gcat.davidson.edu/GcatWiki/index.php/Davidson_Missouri_W/Double_Digest_Guide Buffer Website]

Total volume of enzymes has to be 10% or less than final volume (most volume 2uL). Barely submerge the pipet tip into the solution when pipeting the enzymes.
Star (*) activity: Restriction enzyme stops cutting specifically and starts cutting everywhere.

DNA can't be seen by itself so we add EtBr (the bigger the insert, the more will bind) so bigger strands appear brighter. samples.

The order of entry doesn't matter, but make sure to enter the enzymes last.

Will have lots of labeled tubes and will put 3uL of appropriate plasmids into the tubes.
In the meantime, we'll make a "Master Mix" of all the other ingredients in a bigger tube. Always make enough volume for one extra tube.

Enzymes usually have an optimal temperature of 37C. We will let this reaction go for an hour and then we will run it on a gel.

'''Gel Electrophoresis Protocol'''

Website online for ideal gel concentration for resolving different sizes of molecules [http://gcat.davidson.edu/iGEM08/gelwebsite/gelwebsite.html Gel Concentration Website].
See protocol on the Davidson/Missouri Western Wookie for running molecules in the gel and how to digest with restriction enzymes.

'''What's Next?'''

1. 5' end additions
a. Using PCR
b. Cut at the restriction sites EcoR1 and either Bam H1 or Nco1
c. Cut plasmid and reporter with same restriction enzymes as above
d. Run a gel and purify "keeper" DNA (Ligate transform, screen, sequence)
e. Then, we take the "keeper" DNA and combine it with the insert from part

We can have steps d and e ready in advance.

2. tRNAs
a. Assemble the oligos into genes (Requires lots of calculations beforehand, but the actual steps )
b. Plasmids cut up with EcoR1 and Pst1 '''WE CAN USE THE "WASTE" PLASMID FROM THE RESTRICTION ENZYME DIGEST'''
c. Gel purify the plasmids (Ligate transform, screen, sequence)

The following tables include Master Mix measurements, agarose gel concentrations and buffers.
http://gcat.davidson.edu/GcatWiki/images/0/08/Shamitapic3.png
http://gcat.davidson.edu/GcatWiki/images/d/d1/Shamitapic4.png
http://gcat.davidson.edu/GcatWiki/images/6/6f/Shamitapic5.png

We made the Master Mix with our DNA and put it in the incubator at 10:40 AM.

'''Preparing the Gels'''

Now, we will be making 2 gels, 1 at 0.8% and 2.5% (weight to volume percent). The gel volume is 60mL.

'''Definition of 1% solution:
1 gram of agarose/ 100mL buffer'''

So for a 0.8% solution: 0.8 grams agarose/100mL buffer AND we need 60mL (fixed volume for casts run) so multiply that by 0.6.
Final concentration:

.48 grams agarose/60mL buffer

1 uL of EtBr stock for 60mL of gel.
EtBr is in the buffer so it is constantly replenishing itself even though it is moving toward one end in order to make bands on the other end visible.

After Dr. Campbell's demonstration, we each helped prepare a 0.8% buffer solution using the agarose powder, buffer and EtBr. We mixed together the first two substances in a 200mL Erlenmeyer flask and heated it for 120 seconds in the microwave. We then added EtBr and poured the gel solution into the gel cast.

'''Agar Plate'''

After lunch, we grew some more cells for each of the 11 parts in an agar plate labeled "LBAMP-AMC-9 June" so that so would have extra cells that could be stored for up to 2 weeks.
We made small circles on the agar plate and numbered them 1-11. The numbers corresponded to the following parts. We put the plate in the incubator overnight to let the cells grow.

1. B0030 AMC
2. K091111
3. S03710
4. I715039 OEH
5. S03511 ORH
6. I715039 AST
7. B0030 SDP
8. K091112
9. J31007 LJH
10. J31007 REC
11. S03511

'''Unloading the Gels'''

The following shows which parts we entered in which gels:

[[Image: screen-capture-2.png]]

We unloaded the 2.5% agarose first and after observing the bands under a UV light, put the gel into the BioRad machine to observe the bands. The image below is of a Molecular Weight Marker that shows the number of bp in control fragments of DNA according to the distance they travel down the gel.

[[Image: DNA_molecular_weight_marker_gel.png]]

We obtained the following printout for the first gel that was 2.5% Agarose.
[[Image: 2009-06-09_14hr_44min.jpg]]

The bands in the first well at the bottom are those of the molecular weight marker. We skipped the lane above (to the right) of the MWM and entered the rest of the inserts as described in the above table. A light band is visible for each of the 4 other wells. In Lanes 3 and 4 we concluded we are supposed to have approximately 45 bp because the insert is 15bp + 30 bp on either side of the insert (Xba1 and Spe1 restrictions sites + sticky ends + extra bases). We used the same logic for Lanes 5 and 6 which are supposed to contain approximately 86 base pairs.

One discrepancy in this gel, however, is that Lanes 5 and 6, which according to their labels respectively contain the LacIQ promoter and the Lac IQ1 promoter. According to the information online at the parts registry website, pLacIQ1 should be the same length as pLacIQ. The gels do not show this, but instead show that the sample in Lane 5 is smaller than that in Lane 6 because Lane 5 traveled further. Leland Taylor and Romina Clemente sent out an email to Pallavi Penumetcha to ask about this because she had written a paper about this.

We then unloaded the 0.8% agarose gel and then placed this in the BioRad machine under the same protocol as above. The following image is the 0.8% Agarose.

[[Image:2009-06-09_15hr_21min.jpg]]

We observed that the bands in Lane 1 (which is now at the top) are examples of high intensity bands. The plasmids contained in the original well are twice the intensity of the insert.

In Lane 5, however, we observed several different bands. This could be due to the extra DNA that was sitting on the top of the well. We decided to re-expose the gel to get better observations on Lanes 3 and 4 because the bands were not as intense. We anticipated seeing a band at about 250 bp because this was the size of the insert (plus extra bp). After re-exposing the gel, we did not find any difference and still did not see any bands in these lanes. We have decided to redo the gel on the part S03511 and so we prepared the master mix with that DNA and incubated it so that we could run the gel tomorrow.

We also cut out the "empty" plasmids from the gel so that we could use this as vectors for the coding sequence of the suppressor tRNAs.

Tomorrow we will be planning out specific tasks for people once the oligos arrive.

==Wednesday, June 10, 2009==

We began this morning by making the agarose gel with the same protocol as yesterday. This time, since we are just re-doing both S03511 inserts, we will have a 1.8% agarose gel.

'''OUTLINE: WHERE DO WE STAND?'''

Yesterday
a. Isolated empty plasmids
b. Verified most inserts
c. Waiting for:
>tRNA oligos
>Primers
a. Will amplify the reporter gene in the plasmid itself

Next?
a. When Primers Arrive
> Amplify the reporter genes with primers (PCR takes ~3 hours)
> Calculate how much reporter plasmid we will need (Start with 50x more than we actually will need)
> Cut amplified sequence (EcoR1 and BamH1/Nco1) then purify what we need
> Cut reporter plasmid (EcoR1 and BamH1/Nco1) then purify what we need
> Ligate amplified sequence and reporter plasmid
b. When tRNA oligos arrive
> Calculate how much of each oligo we will need (takes a while...)
> "Boil and cool" tRNA oligos together
> Ligate fragments (dsDNA) into plasmids we isolated yesterday (~5 minutes)
> Transform the plasmids (~20 minutes)

* LOOK THROUGH LIGATION PROTOCOL (How to assemble genes)
* LOOK THROUGH TRANSFORMATION PROTOCOL

''What we need to get done today'':
1. Prepare the control for the Tet Resistance by BioBricking the part pLac+RBS (S03511) with the TetA (J31007).
2. Check the part number for RFP and also for pLac+RBS+RFP
3. Read through ligation protocol
4. See how many nanograms of plasmid we will need for the tRNAs and for the inserts and multiply this number by 50.
> Check if we have enough plasmids from our mini-prep
5. Purify the plasmids from the gel

After we made this outline, Leland, Olivia and I analyzed the gel we ran this morning and did not see a more than one band in the 2 lanes that contained DNA from S03511 (image below). Since we did not see multiple bands in yesterday's gel either, we conclude that there is something wrong with the DNA rather than with the restriction enzymes that we used to cut it. We concluded that there is probably not an insert in this plasmid both the part and the plasmid are useless.

[[Image: 2009-06-10 10hr 30min.jpg]]

Now, we do not have an immediately accessible promoter. We have decided to order the pBAD promoter from Missouri Western and we will attach the RBS+Tet to this plasmid. Our insert for this will be the larger gene because it is easier to manipulate in the gel than the small gene.

Alyndria Thompson and Romina Clemente are figuring out the size of the plasmids including the reporter genes.

After lunch we performed several steps to isolate and purify the gel to get the empty plasmids will be the vectors for the tRNA DNA. Then, we used the NanoDrop to assess the purity of our DNA. We found that the plasmids were more pure than we had expected. The following values were obtained:

Abs 1.850
A-260 10 mm path 0.172
A-280 10 mm path 0.075
260/280 2.28
260/230 0.09

We then followed the protocol for preparing the agar plates. We put the plate with the agar solution in the Autoclave.

While the solution was in the autoclave, we discussed that we don't really need a promoter in front of RBS+Tet for the gene to be expressed because Tet expression is so strong that the gene would translate without a promoter. However, it would be a good idea to have the promoter because if it was possible to reinforce the translation of the reporter protein, we wanted to do that.

We found that there were some tubes in the lab fridge that contained the pBAD and pLac promoters with RBS. We decided to use these as the promoter and RBS for our experiments. The pLac promoter will be used for RFP and the pBad promoter used with Tet Resistance. We decided to use the pBad with Tet because it is weaker in comparison to pLac. As expression of the Tet gene can be toxic to a cell, we do not want to overexpress the gene with a stronger promoter.

Once the agar solution was sterilized in the Autoclave, we removed it, cooled it, and pipetted the antibiotic resistance into it. We then poured the solution into the plates, removed any bubbles, and let the gel form.

Alyndria Thompson and Romina Clemente concluded that we had more than enough recieving vectors for both the RFP and Tetracycline genes. Since we need 50 nanograms, it is wise to have at least 50 times that much that we can work with, so we need at least 2,500 ng.

RFP 1: 77.6 x 4 = 310.4 ng/uL
x 20 uL = 3,272 ng
RFP 2: 59.5 x 4 = 238 ng/uL
x 20 uL = 4,760 ng
Tet: 40.9 x 4 = 163.6 ng/uL
x 20 uL = 3,272 ng

According to these calculations we have more than 50 times what we need for both instances of RFP and Tet.

Tomorrow, we can prepare the Master Mix for the digestions of both reporter plamids and then add the restriction enzymes once they arrive. Then, we can follow through with the rest of the plans we had made above in the section titles "Next?".

==Thursday, June 11, 2009==

We began this morning by calculating how many uL we would need for each of the mixes for digesting our reporter plasmids. We decided to double the final volume of each mix to 40uL instead of 20uL. Since we knew the concentration of each plasmid and also knew we needed 2500 ng, we cross multiplied to find the uL of DNA we would need for each.

Concentration Calculations:
310.4 ng/1 uL = 2500ng/x uL
x = 8.05 uL

Here is a chart indicating how much of each we used for the 2 mixes.

[[Image: DoubledigestRFPTetR.png]]

On the Promega website, we found a list of compatible buffers for double digestions. Buffers E and H are compatible for the Tet plasmid and RFP plasmid digestions respectively. We fortunately have both buffers in stock.

[[Image:BamEcoR1.png]] [[Image:NcoEcoR1.png]]

We waited for our waited for the restriction enzymes to arrive and in the meantime, we transformed the plasmids with pLac+RBS, pBad, and pBad+RBS into ''E.coli'' cells. We want the ''E.coli'' cells to replicate the plasmids with these genes because we have a limited amount of genes. [http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_Transformation.html Zippy Plasmid Transformation]

Once our oligos and restriction enzymes arrived, we got to work right away!

'''PCR (Polymerase Chain Reaction)'''

We calculated how much plasmid we will need for the PCR. We decided to dilute the concentration of our plasmids a hundred fold so that we could get pipetable amounts.

''Change in Concentrations''
310.4 ng/uL x 1/100 (dilution factor) = 3.104 ng/uL (RFP)
163.6 ng/uL x 1/100 (dilution factor) = 0.164 ng/uL (Tet)

''Example shown with RFP''
3.104 ng/1 uL = 1ng/ x uL, x = .32
For Tet, x = .61

We also calculated how much of each primer we would need with the following equation:

C1V1 = C2V2
(100 uM) (x) = (1 uM)( 100 uL)
x = 1 uL

[[Image: screen-capture-8.png]]

Each of the 5 of us made had 2 tubes (one for each reporter protein) because there were 5 different forward primers.To each tube, we added the template DNA (either RFP or Tet), water, the respective reverse primer (either RFP or Tet), and the forward primer depending on which 5mer we had chosen. My 5mer was CCAUC. Finally, we added the Master Mix last because this contained the enzyme (DNA polymerase) that would amplify our desired sequence.

[[Image: Pcrtubes.png]]

I had CCAUC as 5mer.

We followed the temperature cycles so that PCR could occur. The annealing temperature needed to be 5C less than the smallest melting temperature of the primers. Since the reverse primer of RFP had a melting temp of 66C, we set the temperature of the 3rd cycle on 61C.

[[Image: Temperaturecycle.png]]

'''tRNA Oligo Assembly'''

We needed to calculate the concentration of each of the oligo before carrying out the assembly. The stock concentration of each of the oligo was 100 uM. Since we wanted to have 5uM of the oligo in the test tube, and we know that final volume of the test tube is 20 uL, we just cross multiplied to find the amount we needed to add to the tubes to get this concentration.

(100uM)x = (5uM)(20uL)
x = 1 uL

We added 1 uL of each oligo to the tubes.

We had six total tRNAs we needed to assemble so each of us took one FML (one person took two) and to each tube we added 1uL of each oligo for a total of 7uL, 2uL of buffer, and 11uL of water for a total volume of 20 uL. Five of the oligos were constant and 2 varied for each of the different tRNAs. So everyone pipetted the same 5 oligos and everyone also had 2 unique oligos to add. When all of the oligos were added and the tubes ready, we boiled the tubes at 50C for 5 minutes. We then allowed the tubes to remain in the water to cool very slowly overnight so that the annealing process could take place.

The last thing we did at the end of the day was finish off our plasmid digestion that we had begun in the morning. We added the buffer, DNA, water, and restriction enzymes and placed this in the incubator for overnight digestion.

'''To Do''':

1. Isolate our desired dsDNA from the PCR.
2. Verify our tRNA inserts and eventually transform these into plasmids.
3. Check our agar plates to see colonies that have replicated our pLac+RBS, pBAD+RBS, and pBAD genes.

==Friday, June 12, 2009==

This morning, we began by outlining the main items on today's agenda.

1. tRNAs: Ligation, Transformation
2. Digestion reporters (run all of it and gel purify)
3. Transformed cells
4. PCR: Gel run an aliquot to verify (5uL)

We used the equation given by the Ligation protocol to calculate how many nanograms of insert we would need. Initially, we started out calculated the MW average of the 9 and 10 anticodon loops of tRNAs which were 15828.23 ug/umols and 159016.6 ug/umols respectively. We then saw, however, that these would not be helpful values in the process. Instead we started out with the equation below to find the final ng of insert we would need for the ligation.

[[Image: ligationcalculations.png]]

We calculated our initial concentration in ng/uL of insert as shown below.

[[Image: ligationequation.png]]
X nanograms of insert = [(2)(144bp)(50 ng linearalized plasmid)]/2038bp
X = 7.0657 ng of insert

We wanted to find the amount of uL of insert we would need to use. We diluted the initial concentration 50x because we wanted a pipettable amount of insert. Since we knew we wanted the final amount of nanograms to be 7.0657, we calculated as shown below:

''DILUTION'': 555.48/50 = 11.012
''CALCULATION'': 11.012 ng/ 1uL = 7.065 ng/ x uL
x = .64 uL of insert

After we had done these calculations, half of the group started to carryout the ligation of the tRNA inserts into empty plasmids. We needed 7 ligations total: 6 were the tRNAs we needed, while the last one is a negative control. These are the final amounts of what we used:

[[Image: Trnaligation.png]]

The other half of the group (which included me) prepared the agarose gels so that we could run gels for the digested reporters and the PCR inserts. We calculated that the digested reporters needed an agarose of 0.4% and the PCR reporters needed an agarose of 1.45%. We allowed this gel to form for about 45 minutes to an hour. We prepared both the gels and allowed them to run through lunch.
After lunch, we took pictures of our gels and noticed that the third lane in the PCR gel (third lane from the top in the bottom photo) did not develop a band. The lane was supposed to contain the CUAGU insert for Tetracycline Resistance.

[[Image: 2009-06-12 15hr 54min.jpg]] [[Image:2009-06-12_16hr_07min.jpg]]

Romina Clemente and I decided to repeat this PCR using the TetResistance and RBS+TetResistance plasmids as templates. We labeled J31007 as "2*" and S03710 as "2!". We had to recalculate how much template DNA to use. We let the PCR run overnight. We also verified that the digestion fragments (both the plasmid and the insert) on the other gel were the correct size. The plasmids, which are the brighter fragment in the gel with only 2 lanes, contain the plasmid base pairs along with the rest of our reporter genes. We isolated these plasmids from the gel today and stored them for purification on Monday.

In the meantime, other members of our lab had finished the ligation of the tRNA genes to the empty plasmids and were now transforming these into competent cells. They placed the agar plate with these cells in the fridge overnight.

We also observed the transformations that we had performed yesterday and noticed that I13453 did not form any colonies. J04500 and J3102 did form colonies.

To Do:
1. PCR: Dig, Ligate, Transform
Clean and concentration column, digest, clean and concentration column, ligate.
2. Gel purification of our digestion plasmids
3. Check if colonies have grown for our tRNAs

==Saturday, June 13, 2009==

Alyndria Thompson came in on Saturday morning to remove the tRNA plates and put the PCR reactions in the fridge.

'''tRNA Colonies'''

She counted the colonies of tRNA on each plate.

[[Image:TRNA_colonies_6_13_09.png]]

She placed the plates in the large fridge on the top shelf at 9:23 am. She talked to Dr. Campbell who said we will have to try to "work them up".

'''PCR'''

Products were placed in orange rack of smaller fridge. [2*, 2!]

==Sunday, June 14, 2009==

'''Second Ligation and Transformation of tRNA inserts'''

We decided to re-ligate and re-transform all of the tRNA inserts because we did not get very good yields of colonies from the transformations.
We are using the same measurements for this ligation as we had used on Friday.

[[Image: Trnaligation.png]]

After ligation, we transformed the cells. Using the following measurements, we transferred the competent cells into new tubes.

40uL of competent cells
10uL of ligation mixture
60uL of LB media

After spreading the mixture onto the agar plates, we incubated the plates at 10:56 pm.

We also stored liquid cultures of the colonies that greater than a 2:1 variable:negative control ratio just in case we wanted to use them for mini preps in the future. These included #1 (3 samples), #4 (2 samples), #5 (1 sample), and #6 (2 samples).

'''To Do''':
1. Count colonies obtained from second transformation
2. Clean PCRs: Dig, ligate, transform
3. Run gel for 2 Tet PCRs, clean
4. Gel purify receiving vector

IGEM Notebook

2009-06-15T21:52:05Z

ShPunjabi: /* Friday, June 12, 2009 */

==Wednesday, June 3, 2009==

Romina Clemente and I are trying to find suitable reporter proteins to use. Yesterday, Leland Taylor and Alyndria Thompson were working on ways to insert the gene sequences into the plasmid. Upon seeing how they wanted to manipulated the reporter gene to include the logical clauses, we came up with a few criteria for the reporter genes we would use. The following criteria for genes are listed in order of the broadest aspect to look at to the narrowest aspect:
a) Doesn't contain restriction sites for the 4 restriction enzymes (EcoR1, Xbal, Spel, Pst1) used to cleave the Biobrick part out of the plasmid.
b) Contains 6 cutter restriction sites.
c) These restriction enzymes aren't blunt (cleave straight down at one spot).
d) These restriction sites are close to thge 5' (beginning) end of the sequence.
e) These enzymes are easiest to work with and cheapest.
We are finding the part numbers of the reporter genes we want to use (antibiotic resistance, fluorescence, LacZ) through our own GCAT because we know these ones work. We are then locating these parts on the parts registry [1] website. We copied and pasted the gene sequences we obtained from the registry onto the ApE software [2]. From here, we were able to generate a genetic map of each gene that outlined each restriction site that fit our criteria. We put each genetic map, alongside the part number used, into a Word document.

http://gcat.davidson.edu/GcatWiki/images/0/0e/Restriction_Site_Mapping_on_Reporter_Genes.doc

Later on in the day, we decided that the best way to test for suppression would involve placing the 5mers at the beginning of the reporter gene instead of inserting them into the reporter gene. To do this, we decided that the gene along with its start codon need to be expressed after the 5mer. We will insert a BioBrick plasmid with a 5mer and start codon that will be incorporated before the gene:
RBS-6-8nt-ATG-5mer--ATG-gene

Leland Taylor assembled the oligos that we will need for the BioBrick pieces.

In order to test whether the reporter protein would be expressed regardless of the 5mer being suppressed or not, we would need to remove the suppressor tRNAs from the cell.

Meanwhile, I helped figure out the coding sequence that we will need for the suppressor tRNAs that will bind to the 5mers. We decided that we would be using the following suppressor tRNAs on the Davidson side: CUAGU, CCCUC, CGGUC, CCAUC, and CCACU. We searched through several papers, with little result and finally emailed Dr. Christopher Anderson of UC Berkeley to request the DNA sequence. He emailed us back with a generic DNA code that we could use to create the tRNA; however, for each suppressor tRNA we would use, we would need to change base pairs in the anticodon loop.

The length of the tRNA is 92 nt. The anticodon loop is comprised of 9 nt. Once we have BioBricked the DNA sequence with 4 restriction sites and supplemental nucleotides, the length of the entire gene is 144 nucleotides. This is made up of the 92 nt +22 nt on each side of the sequence (44) + 4 nt on each side of the restriction ends (8).

At the end of the day, we decided to use tetracycline resistance and RFP as our reporter proteins for this tester experiment.

==Thursday, June 4, 2009==

This morning, I began the day by learning how to use the PEARL prgram. Olivia Ho-Shing sat with me and walked me through the program script. And then, Leland Taylor and Shashank Suresh had a problem they wanted us to solve. Leland explained that we need to make sure there is a stop codon in the gene if we fail to suppress the 5mer. The stop codon needs to be before we find another 5mer. They also wanted us to run through both the RFP and Tet Resistance genes to make sure that they did not contain any of the anticodon sequences from our 5 frameshift suppressor tRNAs so as to throw the translation out of frame. They further wanted us to confirm that if we do have successful suppression, there are no stop codons in the middle of the RFP and Tet Resistance genes. PEARL did not find any matches for the suppressor tRNAs within genes. We found 3 matches for stop codons UAA. Two of these occured at the end of the Tet and one at the end of RFP, where they should normally be to end translation into a protein.

Following this, Olivia Ho-Shing and I tried to determine how we can add BioBrick ends to our single stranded DNA sequences that code for the suppressor tRNAs. Our ultimate goal is to find complements for these single strands so they can be put into plasmids. We found the standard prefixes and suffixes for sequences that do not contain ATG (becuase this sequence is a functional RNA and will not be translated) and we placed these before and after the altered 92bp sequence that Dr. Anderson provided us with. The prefixes and suffixes are necessary because they are the extra nucleotides that will be mimicking a "restriction enzyme cutting site". The single strand appeared as below after the Bio Brick prefixes and suffixes were added:

22ntPREFIX--32nt--VARIABLE ANTICODON LOOP--51nt--21ntSUFFIX

We used the suppressor codon CUAGU as our example and placed its appropriate anticodon loop in the sequence. We put this into the lancelator that would give us the other strand as well as the oligos we would need for ideal construction of this double stranded DNA fragment to put into the plasmid. We recieved a total of 4 oligos, 2 of which were variable and 2 of which were constant for all 6 different suppressor tRNAs. Using this double strand, we simulated a restriction enzyme digestion by EcoRI and PstI. We took off the G at both ends of the top strand and then removed the first five nucleotides on each end of the bottom strand. This made the top strand longer than the bottom one. We then proceeded to take the two variable strands and changed the anticodon loop nt for each suppressor tRNA we used.

Around lunchtime, we encountered a problem with the BioBrick scar in the "ATG-5mer subpiece" of the test project. It turns out that after annealing the sticky ends of the Xba1 and Spe1 sites, the scar created had a stop codon TAG (UAG) that was in frame with the suppressed codon. When the Xba1 site on insert anneals to the Spe1 site on the plasmid (the green portions), we get a scar that reads TACTAG.
[[Image:screen-capture.png]]

For the afternoon, Olivia Ho-Shing and I finished assembly of the oligos of the suppressor tRNA codes. We assembled the document below:

http://gcat.davidson.edu/GcatWiki/images/5/5e/TRNAoligostoOrder.doc

We were assigned tasks of creating "cartoons" to illustrate the phenomenon that had occurred. No one could successfully do this however, because we could not understand how the piece had formed. The parts registry website provided wrong information on the restriction site of Spe1 which slowed our efficiency. This project turned into an effort to try to find two other different restriction enzymes that have complementary sticky ends other than Xba1 and Spe1. We want to get rid oc the scar that contains TACTAG.

We ended up deciding that a hybrid of two ideas was needed: restriction enzymes would need to be used to make several reporter gene sequences with different beginnings dependending on the suppressor tRNA sequence needed.

==Friday, June 5, 2009==

Our goal for this morning was to discuss the hybrid PCR approach to assemble the part that comes before the gene we wanted to express. We decided that we would have a BioBrick prefix and a 5mer followed by a sequence of nucleotides whose identity was up to us. Using PCR we would get the second double strand if we have the following:

BioBrick Prefix--ATG--5mer--gene until we get to restriction site

The PCR primer would attach to the ATG and the 5mer and would replicate the complementary base pairs to make a double stranded DNA sequence. As several of us were confused about PCR methods, we looked at the online link provided by Alyndria Thompson's link to the PCR interactive website which was very helpful. We also did some research to discover that the restriction enzyme BseRI (which is contained in the YFP that we wanted to alter) is not compatible with any other restriction enzyme. This is good because we did not want to plasmid containing this gene to close up with Xba1 once we digested it.

The team experienced some confusion with the hybrid idea and spoke to Clif Davis of Missouri Western University on the phone. He told us that there will be one part that is a fragment of the beginning of some nucleotides as a restriction cleaving site, ATG, the logical clause, and the start of the YFP gene that will be placed into a plasmid containing a space. To the left of the space will be the BioBrick Prefix and the right of the space will contain the rest of the genes in the YFP. Therefore, we decided not to alter any BioBrick ends and just look at restriction sites at the beginning of reporter proteins. The small fragment that will be placed into the plasmid will be made by PCR. The PCR will attach a primer onto the ATG and 5mer and replicate.

While we were discussing this, we expanded upon the idea that we can do whatever we want with the "suffix" portion between the 5mer and the beginning of YFP. We decided it would be best to remove ATG and add the first 5 amino acids and the restriction enzyme site. Along with this, if we have the 5mer coding for a certain amino acid (ie methionine) we could replace another codon that codes for methionine WITH that 5mer so long as this codon is contained within the first 15 nt. This way, we would not be altering the protein.

Olivia Ho-Shing came up with an idea of cascading PCR effect for longer logical clauses. Post translational modification of the protein was another one of her ideas. This applies because although we may code for the protein in a given way, the protein may modify or fix itself up to perform a slightly different function in its tertiary form.

Dr. Campbell suggested that we can look up restriction enzymes 400-500 base pairs into the reporter protein we would like to use; however, we have more flexibility in our procedure if we choose more "hardy" cutters. So instead, we could cut with EcoRI instead of with Xba1.

During the afternoon, we set about three tasks:
1) Which primers to use
2) Which promoters to use
3) Which restriction enzymes and how they will determine use of reporter proteins

Olivia Ho-Shing and I began looking up which restriction enzymes were strong so we could base our reporters on that. We began with Tet and RFP since those were the two reporters we had started out with choosing. I found a website that provided enzyme cleavage efficiency based upon the proximity of the site to the end of the PCR fragment that contains it. We discovered the following based upon the attached documents:

http://gcat.davidson.edu/GcatWiki/images/8/8f/Restriction_Enzyme_Cleavage_Efficiency.doc
http://gcat.davidson.edu/GcatWiki/images/9/9a/07Jun09_RE_Cutting_Efficiency_oh.doc

We decided that these two reporters contained efficient enzymes in a good quantity to choose from. On Monday, we will look through these enzymes once more and finalize which enzymes we would like to use with RFP and Tet.

==Sunday, June 7, 2009==

We did preparations for the mini prep this evening. The following notes are courtesy of Olivia Ho-Shing.

1. Sterically transfer ~2mL LB+AMP (AMP are ampicillin resistant plasmids) *Never put fluid back into bottle once it has entered pipette*
2. Use forceps to grab toothpick, scrap some frozen cells, rub in tube with top

'''Tubes'''

S03511 (2)

K091111

B0030

I715039

K091112

I715039

J31007 (2)

B0034

E1010

S03710

==Monday, June 8, 2009==

We reviewed the PCR process this morning as well as the restriction enzyme cleavage process.

Then, Leland Taylor, Olivia Ho-Shing and I all worked on converting the tRNA oligos we made to the template that Missouri Western used. The oligos we had previously made were different from the ones that Dr. Anderson had originally emailed us because further communication between Dr. Anderson and Missouri Western resulted in a better template.

We tried several times to minimize the number of oligos while keeping their individual lengths under 80bp. We managed to do this in the end by creating 7 oligos, 2 of which are variable between the suppressor tRNAs.
We reviewed the following document and finalized the oligos that we needed to order for the frameshift suppressor tRNAs. We decided to keep both the 10bp and 9bp anticodons of CCAUC.

I also looked up the amino acids that corresponded to the FS (frameshift suppressor) tRNAs for future use:

1. CUAGU = Leu
2. CCCUC = Pro
3. CGGUC = Arg
4. CCACU = Pro
5. CCAUC* = Pro
*9 and 10 bp anticodons

Also, we have decided to use NcoI as the restriction enzyme for RFP. This cleavage site occurs 419 bp into the gene. We have decided to use BamHI as the enzyme for Tetracycline Resistance and the cleavage site occurs 290bp into the gene. Both of these enzymes are very powerful and hardy.

We also looked over Missouri Western's tRNA oligos and primer oligos which we approved.
Our oligos were also approved by Missouri Western and Dr. Campbell ordered them while we supervised.

In the afternoon we did the miniprep. The mini prep directions were as appear in the link below.

http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_MiniPrep.html

We then worked with the Nano Prep to dilute our solution of DNA to an absorption of 1.5 and then we made labels for the tubes. The chart below shows relevant information we obtained from the NanoDrop for each of the reporter plasmids.

[[Image: Screen-capture-6.png]]

==Tuesday, June 9, 2009==

'''Preparations for restriction enzymes digestion of plasmids'''

Purpose: To determine the size of the plasmid inserts and ensure that plasmids are what we extracted from the cells during the mini-prep.

Things we need:
DNA ~3uL
Buffer 10x (Want to dilute to 1x) so add 2uL
Enzyme 1 ~1uL
Enzme 2 ~1uL
Water ~13uL
Final Volunter about 20 uL.

Need to choose the right buffer for the restriction enzymes. All buffers come in 10x (10 times more concentrated than what you want it to be when you use it). [http://gcat.davidson.edu/GcatWiki/index.php/Davidson_Missouri_W/Double_Digest_Guide Buffer Website]

Total volume of enzymes has to be 10% or less than final volume (most volume 2uL). Barely submerge the pipet tip into the solution when pipeting the enzymes.
Star (*) activity: Restriction enzyme stops cutting specifically and starts cutting everywhere.

DNA can't be seen by itself so we add EtBr (the bigger the insert, the more will bind) so bigger strands appear brighter. samples.

The order of entry doesn't matter, but make sure to enter the enzymes last.

Will have lots of labeled tubes and will put 3uL of appropriate plasmids into the tubes.
In the meantime, we'll make a "Master Mix" of all the other ingredients in a bigger tube. Always make enough volume for one extra tube.

Enzymes usually have an optimal temperature of 37C. We will let this reaction go for an hour and then we will run it on a gel.

'''Gel Electrophoresis Protocol'''

Website online for ideal gel concentration for resolving different sizes of molecules [http://gcat.davidson.edu/iGEM08/gelwebsite/gelwebsite.html Gel Concentration Website].
See protocol on the Davidson/Missouri Western Wookie for running molecules in the gel and how to digest with restriction enzymes.

'''What's Next?'''

1. 5' end additions
a. Using PCR
b. Cut at the restriction sites EcoR1 and either Bam H1 or Nco1
c. Cut plasmid and reporter with same restriction enzymes as above
d. Run a gel and purify "keeper" DNA (Ligate transform, screen, sequence)
e. Then, we take the "keeper" DNA and combine it with the insert from part

We can have steps d and e ready in advance.

2. tRNAs
a. Assemble the oligos into genes (Requires lots of calculations beforehand, but the actual steps )
b. Plasmids cut up with EcoR1 and Pst1 '''WE CAN USE THE "WASTE" PLASMID FROM THE RESTRICTION ENZYME DIGEST'''
c. Gel purify the plasmids (Ligate transform, screen, sequence)

The following tables include Master Mix measurements, agarose gel concentrations and buffers.
http://gcat.davidson.edu/GcatWiki/images/0/08/Shamitapic3.png
http://gcat.davidson.edu/GcatWiki/images/d/d1/Shamitapic4.png
http://gcat.davidson.edu/GcatWiki/images/6/6f/Shamitapic5.png

We made the Master Mix with our DNA and put it in the incubator at 10:40 AM.

'''Preparing the Gels'''

Now, we will be making 2 gels, 1 at 0.8% and 2.5% (weight to volume percent). The gel volume is 60mL.

'''Definition of 1% solution:
1 gram of agarose/ 100mL buffer'''

So for a 0.8% solution: 0.8 grams agarose/100mL buffer AND we need 60mL (fixed volume for casts run) so multiply that by 0.6.
Final concentration:

.48 grams agarose/60mL buffer

1 uL of EtBr stock for 60mL of gel.
EtBr is in the buffer so it is constantly replenishing itself even though it is moving toward one end in order to make bands on the other end visible.

After Dr. Campbell's demonstration, we each helped prepare a 0.8% buffer solution using the agarose powder, buffer and EtBr. We mixed together the first two substances in a 200mL Erlenmeyer flask and heated it for 120 seconds in the microwave. We then added EtBr and poured the gel solution into the gel cast.

'''Agar Plate'''

After lunch, we grew some more cells for each of the 11 parts in an agar plate labeled "LBAMP-AMC-9 June" so that so would have extra cells that could be stored for up to 2 weeks.
We made small circles on the agar plate and numbered them 1-11. The numbers corresponded to the following parts. We put the plate in the incubator overnight to let the cells grow.

1. B0030 AMC
2. K091111
3. S03710
4. I715039 OEH
5. S03511 ORH
6. I715039 AST
7. B0030 SDP
8. K091112
9. J31007 LJH
10. J31007 REC
11. S03511

'''Unloading the Gels'''

The following shows which parts we entered in which gels:

[[Image: screen-capture-2.png]]

We unloaded the 2.5% agarose first and after observing the bands under a UV light, put the gel into the BioRad machine to observe the bands. The image below is of a Molecular Weight Marker that shows the number of bp in control fragments of DNA according to the distance they travel down the gel.

[[Image: DNA_molecular_weight_marker_gel.png]]

We obtained the following printout for the first gel that was 2.5% Agarose.
[[Image: 2009-06-09_14hr_44min.jpg]]

The bands in the first well at the bottom are those of the molecular weight marker. We skipped the lane above (to the right) of the MWM and entered the rest of the inserts as described in the above table. A light band is visible for each of the 4 other wells. In Lanes 3 and 4 we concluded we are supposed to have approximately 45 bp because the insert is 15bp + 30 bp on either side of the insert (Xba1 and Spe1 restrictions sites + sticky ends + extra bases). We used the same logic for Lanes 5 and 6 which are supposed to contain approximately 86 base pairs.

One discrepancy in this gel, however, is that Lanes 5 and 6, which according to their labels respectively contain the LacIQ promoter and the Lac IQ1 promoter. According to the information online at the parts registry website, pLacIQ1 should be the same length as pLacIQ. The gels do not show this, but instead show that the sample in Lane 5 is smaller than that in Lane 6 because Lane 5 traveled further. Leland Taylor and Romina Clemente sent out an email to Pallavi Penumetcha to ask about this because she had written a paper about this.

We then unloaded the 0.8% agarose gel and then placed this in the BioRad machine under the same protocol as above. The following image is the 0.8% Agarose.

[[Image:2009-06-09_15hr_21min.jpg]]

We observed that the bands in Lane 1 (which is now at the top) are examples of high intensity bands. The plasmids contained in the original well are twice the intensity of the insert.

In Lane 5, however, we observed several different bands. This could be due to the extra DNA that was sitting on the top of the well. We decided to re-expose the gel to get better observations on Lanes 3 and 4 because the bands were not as intense. We anticipated seeing a band at about 250 bp because this was the size of the insert (plus extra bp). After re-exposing the gel, we did not find any difference and still did not see any bands in these lanes. We have decided to redo the gel on the part S03511 and so we prepared the master mix with that DNA and incubated it so that we could run the gel tomorrow.

We also cut out the "empty" plasmids from the gel so that we could use this as vectors for the coding sequence of the suppressor tRNAs.

Tomorrow we will be planning out specific tasks for people once the oligos arrive.

==Wednesday, June 10, 2009==

We began this morning by making the agarose gel with the same protocol as yesterday. This time, since we are just re-doing both S03511 inserts, we will have a 1.8% agarose gel.

'''OUTLINE: WHERE DO WE STAND?'''

Yesterday
a. Isolated empty plasmids
b. Verified most inserts
c. Waiting for:
>tRNA oligos
>Primers
a. Will amplify the reporter gene in the plasmid itself

Next?
a. When Primers Arrive
> Amplify the reporter genes with primers (PCR takes ~3 hours)
> Calculate how much reporter plasmid we will need (Start with 50x more than we actually will need)
> Cut amplified sequence (EcoR1 and BamH1/Nco1) then purify what we need
> Cut reporter plasmid (EcoR1 and BamH1/Nco1) then purify what we need
> Ligate amplified sequence and reporter plasmid
b. When tRNA oligos arrive
> Calculate how much of each oligo we will need (takes a while...)
> "Boil and cool" tRNA oligos together
> Ligate fragments (dsDNA) into plasmids we isolated yesterday (~5 minutes)
> Transform the plasmids (~20 minutes)

* LOOK THROUGH LIGATION PROTOCOL (How to assemble genes)
* LOOK THROUGH TRANSFORMATION PROTOCOL

''What we need to get done today'':
1. Prepare the control for the Tet Resistance by BioBricking the part pLac+RBS (S03511) with the TetA (J31007).
2. Check the part number for RFP and also for pLac+RBS+RFP
3. Read through ligation protocol
4. See how many nanograms of plasmid we will need for the tRNAs and for the inserts and multiply this number by 50.
> Check if we have enough plasmids from our mini-prep
5. Purify the plasmids from the gel

After we made this outline, Leland, Olivia and I analyzed the gel we ran this morning and did not see a more than one band in the 2 lanes that contained DNA from S03511 (image below). Since we did not see multiple bands in yesterday's gel either, we conclude that there is something wrong with the DNA rather than with the restriction enzymes that we used to cut it. We concluded that there is probably not an insert in this plasmid both the part and the plasmid are useless.

[[Image: 2009-06-10 10hr 30min.jpg]]

Now, we do not have an immediately accessible promoter. We have decided to order the pBAD promoter from Missouri Western and we will attach the RBS+Tet to this plasmid. Our insert for this will be the larger gene because it is easier to manipulate in the gel than the small gene.

Alyndria Thompson and Romina Clemente are figuring out the size of the plasmids including the reporter genes.

After lunch we performed several steps to isolate and purify the gel to get the empty plasmids will be the vectors for the tRNA DNA. Then, we used the NanoDrop to assess the purity of our DNA. We found that the plasmids were more pure than we had expected. The following values were obtained:

Abs 1.850
A-260 10 mm path 0.172
A-280 10 mm path 0.075
260/280 2.28
260/230 0.09

We then followed the protocol for preparing the agar plates. We put the plate with the agar solution in the Autoclave.

While the solution was in the autoclave, we discussed that we don't really need a promoter in front of RBS+Tet for the gene to be expressed because Tet expression is so strong that the gene would translate without a promoter. However, it would be a good idea to have the promoter because if it was possible to reinforce the translation of the reporter protein, we wanted to do that.

We found that there were some tubes in the lab fridge that contained the pBAD and pLac promoters with RBS. We decided to use these as the promoter and RBS for our experiments. The pLac promoter will be used for RFP and the pBad promoter used with Tet Resistance. We decided to use the pBad with Tet because it is weaker in comparison to pLac. As expression of the Tet gene can be toxic to a cell, we do not want to overexpress the gene with a stronger promoter.

Once the agar solution was sterilized in the Autoclave, we removed it, cooled it, and pipetted the antibiotic resistance into it. We then poured the solution into the plates, removed any bubbles, and let the gel form.

Alyndria Thompson and Romina Clemente concluded that we had more than enough recieving vectors for both the RFP and Tetracycline genes. Since we need 50 nanograms, it is wise to have at least 50 times that much that we can work with, so we need at least 2,500 ng.

RFP 1: 77.6 x 4 = 310.4 ng/uL
x 20 uL = 3,272 ng
RFP 2: 59.5 x 4 = 238 ng/uL
x 20 uL = 4,760 ng
Tet: 40.9 x 4 = 163.6 ng/uL
x 20 uL = 3,272 ng

According to these calculations we have more than 50 times what we need for both instances of RFP and Tet.

Tomorrow, we can prepare the Master Mix for the digestions of both reporter plamids and then add the restriction enzymes once they arrive. Then, we can follow through with the rest of the plans we had made above in the section titles "Next?".

==Thursday, June 11, 2009==

We began this morning by calculating how many uL we would need for each of the mixes for digesting our reporter plasmids. We decided to double the final volume of each mix to 40uL instead of 20uL. Since we knew the concentration of each plasmid and also knew we needed 2500 ng, we cross multiplied to find the uL of DNA we would need for each.

Concentration Calculations:
310.4 ng/1 uL = 2500ng/x uL
x = 8.05 uL

Here is a chart indicating how much of each we used for the 2 mixes.

[[Image: DoubledigestRFPTetR.png]]

On the Promega website, we found a list of compatible buffers for double digestions. Buffers E and H are compatible for the Tet plasmid and RFP plasmid digestions respectively. We fortunately have both buffers in stock.

[[Image:BamEcoR1.png]] [[Image:NcoEcoR1.png]]

We waited for our waited for the restriction enzymes to arrive and in the meantime, we transformed the plasmids with pLac+RBS, pBad, and pBad+RBS into ''E.coli'' cells. We want the ''E.coli'' cells to replicate the plasmids with these genes because we have a limited amount of genes. [http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_Transformation.html Zippy Plasmid Transformation]

Once our oligos and restriction enzymes arrived, we got to work right away!

'''PCR (Polymerase Chain Reaction)'''

We calculated how much plasmid we will need for the PCR. We decided to dilute the concentration of our plasmids a hundred fold so that we could get pipetable amounts.

''Change in Concentrations''
310.4 ng/uL x 1/100 (dilution factor) = 3.104 ng/uL (RFP)
163.6 ng/uL x 1/100 (dilution factor) = 0.164 ng/uL (Tet)

''Example shown with RFP''
3.104 ng/1 uL = 1ng/ x uL, x = .32
For Tet, x = .61

We also calculated how much of each primer we would need with the following equation:

C1V1 = C2V2
(100 uM) (x) = (1 uM)( 100 uL)
x = 1 uL

[[Image: screen-capture-8.png]]

Each of the 5 of us made had 2 tubes (one for each reporter protein) because there were 5 different forward primers.To each tube, we added the template DNA (either RFP or Tet), water, the respective reverse primer (either RFP or Tet), and the forward primer depending on which 5mer we had chosen. My 5mer was CCAUC. Finally, we added the Master Mix last because this contained the enzyme (DNA polymerase) that would amplify our desired sequence.

[[Image: Pcrtubes.png]]

I had

We followed the temperature cycles so that PCR could occur. The annealing temperature needed to be 5C less than the smallest melting temperature of the primers. Since the reverse primer of RFP had a melting temp of 66C, we set the temperature of the 3rd cycle on 61C.

[[Image: Temperaturecycle.png]]

'''tRNA Oligo Assembly'''

We needed to calculate the concentration of each of the oligo before carrying out the assembly. The stock concentration of each of the oligo was 100 uM. Since we wanted to have 5uM of the oligo in the test tube, and we know that final volume of the test tube is 20 uL, we just cross multiplied to find the amount we needed to add to the tubes to get this concentration.

(100uM)x = (5uM)(20uL)
x = 1 uL

We added 1 uL of each oligo to the tubes.

We had six total tRNAs we needed to assemble so each of us took one FML (one person took two) and to each tube we added 1uL of each oligo for a total of 7uL, 2uL of buffer, and 11uL of water for a total volume of 20 uL. Five of the oligos were constant and 2 varied for each of the different tRNAs. So everyone pipetted the same 5 oligos and everyone also had 2 unique oligos to add. When all of the oligos were added and the tubes ready, we boiled the tubes at 50C for 5 minutes. We then allowed the tubes to remain in the water to cool very slowly overnight so that the annealing process could take place.

The last thing we did at the end of the day was finish off our plasmid digestion that we had begun in the morning. We added the buffer, DNA, water, and restriction enzymes and placed this in the incubator for overnight digestion.

'''To Do''':

1. Isolate our desired dsDNA from the PCR.
2. Verify our tRNA inserts and eventually transform these into plasmids.
3. Check our agar plates to see colonies that have replicated our pLac+RBS, pBAD+RBS, and pBAD genes.

==Friday, June 12, 2009==

This morning, we began by outlining the main items on today's agenda.

1. tRNAs: Ligation, Transformation
2. Digestion reporters (run all of it and gel purify)
3. Transformed cells
4. PCR: Gel run an aliquot to verify (5uL)

We used the equation given by the Ligation protocol to calculate how many nanograms of insert we would need. Initially, we started out calculated the MW average of the 9 and 10 anticodon loops of tRNAs which were 15828.23 ug/umols and 159016.6 ug/umols respectively. We then saw, however, that these would not be helpful values in the process. Instead we started out with the equation below to find the final ng of insert we would need for the ligation.

[[Image: ligationcalculations.png]]

We calculated our initial concentration in ng/uL of insert as shown below.

[[Image: ligationequation.png]]
X nanograms of insert = [(2)(144bp)(50 ng linearalized plasmid)]/2038bp
X = 7.0657 ng of insert

We wanted to find the amount of uL of insert we would need to use. We diluted the initial concentration 50x because we wanted a pipettable amount of insert. Since we knew we wanted the final amount of nanograms to be 7.0657, we calculated as shown below:

''DILUTION'': 555.48/50 = 11.012
''CALCULATION'': 11.012 ng/ 1uL = 7.065 ng/ x uL
x = .64 uL of insert

After we had done these calculations, half of the group started to carryout the ligation of the tRNA inserts into empty plasmids. We needed 7 ligations total: 6 were the tRNAs we needed, while the last one is a negative control. These are the final amounts of what we used:

[[Image: Trnaligation.png]]

The other half of the group (which included me) prepared the agarose gels so that we could run gels for the digested reporters and the PCR inserts. We calculated that the digested reporters needed an agarose of 0.4% and the PCR reporters needed an agarose of 1.45%. We allowed this gel to form for about 45 minutes to an hour. We prepared both the gels and allowed them to run through lunch.
After lunch, we took pictures of our gels and noticed that the third lane in the PCR gel (third lane from the top in the bottom photo) did not develop a band. The lane was supposed to contain the CUAGU insert for Tetracycline Resistance.

[[Image: 2009-06-12 15hr 54min.jpg]] [[Image:2009-06-12_16hr_07min.jpg]]

Romina Clemente and I decided to repeat this PCR using the TetResistance and RBS+TetResistance plasmids as templates. We labeled J31007 as "2*" and S03710 as "2!". We had to recalculate how much template DNA to use. We let the PCR run overnight. We also verified that the digestion fragments (both the plasmid and the insert) on the other gel were the correct size. The plasmids, which are the brighter fragment in the gel with only 2 lanes, contain the plasmid base pairs along with the rest of our reporter genes. We isolated these plasmids from the gel today and stored them for purification on Monday.

In the meantime, other members of our lab had finished the ligation of the tRNA genes to the empty plasmids and were now transforming these into competent cells. They placed the agar plate with these cells in the fridge overnight.

We also observed the transformations that we had performed yesterday and noticed that I13453 did not form any colonies. J04500 and J3102 did form colonies.

To Do:
1. PCR: Dig, Ligate, Transform
Clean and concentration column, digest, clean and concentration column, ligate.
2. Gel purification of our digestion plasmids
3. Check if colonies have grown for our tRNAs

==Saturday, June 13, 2009==

Alyndria Thompson came in on Saturday morning to remove the tRNA plates and put the PCR reactions in the fridge.

'''tRNA Colonies'''

She counted the colonies of tRNA on each plate.

[[Image:TRNA_colonies_6_13_09.png]]

She placed the plates in the large fridge on the top shelf at 9:23 am. She talked to Dr. Campbell who said we will have to try to "work them up".

'''PCR'''

Products were placed in orange rack of smaller fridge. [2*, 2!]

==Sunday, June 14, 2009==

'''Second Ligation and Transformation of tRNA inserts'''

We decided to re-ligate and re-transform all of the tRNA inserts because we did not get very good yields of colonies from the transformations.
We are using the same measurements for this ligation as we had used on Friday.

[[Image: Trnaligation.png]]

After ligation, we transformed the cells. Using the following measurements, we transferred the competent cells into new tubes.

40uL of competent cells
10uL of ligation mixture
60uL of LB media

After spreading the mixture onto the agar plates, we incubated the plates at 10:56 pm.

We also stored liquid cultures of the colonies that greater than a 2:1 variable:negative control ratio just in case we wanted to use them for mini preps in the future. These included #1 (3 samples), #4 (2 samples), #5 (1 sample), and #6 (2 samples).

'''To Do''':
1. Count colonies obtained from second transformation
2. Clean PCRs: Dig, ligate, transform
3. Run gel for 2 Tet PCRs, clean
4. Gel purify receiving vector

IGEM Notebook

2009-06-15T13:09:25Z

ShPunjabi: /* Thursday, June 11, 2009 */

==Wednesday, June 3, 2009==

Romina Clemente and I are trying to find suitable reporter proteins to use. Yesterday, Leland Taylor and Alyndria Thompson were working on ways to insert the gene sequences into the plasmid. Upon seeing how they wanted to manipulated the reporter gene to include the logical clauses, we came up with a few criteria for the reporter genes we would use. The following criteria for genes are listed in order of the broadest aspect to look at to the narrowest aspect:
a) Doesn't contain restriction sites for the 4 restriction enzymes (EcoR1, Xbal, Spel, Pst1) used to cleave the Biobrick part out of the plasmid.
b) Contains 6 cutter restriction sites.
c) These restriction enzymes aren't blunt (cleave straight down at one spot).
d) These restriction sites are close to thge 5' (beginning) end of the sequence.
e) These enzymes are easiest to work with and cheapest.
We are finding the part numbers of the reporter genes we want to use (antibiotic resistance, fluorescence, LacZ) through our own GCAT because we know these ones work. We are then locating these parts on the parts registry [1] website. We copied and pasted the gene sequences we obtained from the registry onto the ApE software [2]. From here, we were able to generate a genetic map of each gene that outlined each restriction site that fit our criteria. We put each genetic map, alongside the part number used, into a Word document.

http://gcat.davidson.edu/GcatWiki/images/0/0e/Restriction_Site_Mapping_on_Reporter_Genes.doc

Later on in the day, we decided that the best way to test for suppression would involve placing the 5mers at the beginning of the reporter gene instead of inserting them into the reporter gene. To do this, we decided that the gene along with its start codon need to be expressed after the 5mer. We will insert a BioBrick plasmid with a 5mer and start codon that will be incorporated before the gene:
RBS-6-8nt-ATG-5mer--ATG-gene

Leland Taylor assembled the oligos that we will need for the BioBrick pieces.

In order to test whether the reporter protein would be expressed regardless of the 5mer being suppressed or not, we would need to remove the suppressor tRNAs from the cell.

Meanwhile, I helped figure out the coding sequence that we will need for the suppressor tRNAs that will bind to the 5mers. We decided that we would be using the following suppressor tRNAs on the Davidson side: CUAGU, CCCUC, CGGUC, CCAUC, and CCACU. We searched through several papers, with little result and finally emailed Dr. Christopher Anderson of UC Berkeley to request the DNA sequence. He emailed us back with a generic DNA code that we could use to create the tRNA; however, for each suppressor tRNA we would use, we would need to change base pairs in the anticodon loop.

The length of the tRNA is 92 nt. The anticodon loop is comprised of 9 nt. Once we have BioBricked the DNA sequence with 4 restriction sites and supplemental nucleotides, the length of the entire gene is 144 nucleotides. This is made up of the 92 nt +22 nt on each side of the sequence (44) + 4 nt on each side of the restriction ends (8).

At the end of the day, we decided to use tetracycline resistance and RFP as our reporter proteins for this tester experiment.

==Thursday, June 4, 2009==

This morning, I began the day by learning how to use the PEARL prgram. Olivia Ho-Shing sat with me and walked me through the program script. And then, Leland Taylor and Shashank Suresh had a problem they wanted us to solve. Leland explained that we need to make sure there is a stop codon in the gene if we fail to suppress the 5mer. The stop codon needs to be before we find another 5mer. They also wanted us to run through both the RFP and Tet Resistance genes to make sure that they did not contain any of the anticodon sequences from our 5 frameshift suppressor tRNAs so as to throw the translation out of frame. They further wanted us to confirm that if we do have successful suppression, there are no stop codons in the middle of the RFP and Tet Resistance genes. PEARL did not find any matches for the suppressor tRNAs within genes. We found 3 matches for stop codons UAA. Two of these occured at the end of the Tet and one at the end of RFP, where they should normally be to end translation into a protein.

Following this, Olivia Ho-Shing and I tried to determine how we can add BioBrick ends to our single stranded DNA sequences that code for the suppressor tRNAs. Our ultimate goal is to find complements for these single strands so they can be put into plasmids. We found the standard prefixes and suffixes for sequences that do not contain ATG (becuase this sequence is a functional RNA and will not be translated) and we placed these before and after the altered 92bp sequence that Dr. Anderson provided us with. The prefixes and suffixes are necessary because they are the extra nucleotides that will be mimicking a "restriction enzyme cutting site". The single strand appeared as below after the Bio Brick prefixes and suffixes were added:

22ntPREFIX--32nt--VARIABLE ANTICODON LOOP--51nt--21ntSUFFIX

We used the suppressor codon CUAGU as our example and placed its appropriate anticodon loop in the sequence. We put this into the lancelator that would give us the other strand as well as the oligos we would need for ideal construction of this double stranded DNA fragment to put into the plasmid. We recieved a total of 4 oligos, 2 of which were variable and 2 of which were constant for all 6 different suppressor tRNAs. Using this double strand, we simulated a restriction enzyme digestion by EcoRI and PstI. We took off the G at both ends of the top strand and then removed the first five nucleotides on each end of the bottom strand. This made the top strand longer than the bottom one. We then proceeded to take the two variable strands and changed the anticodon loop nt for each suppressor tRNA we used.

Around lunchtime, we encountered a problem with the BioBrick scar in the "ATG-5mer subpiece" of the test project. It turns out that after annealing the sticky ends of the Xba1 and Spe1 sites, the scar created had a stop codon TAG (UAG) that was in frame with the suppressed codon. When the Xba1 site on insert anneals to the Spe1 site on the plasmid (the green portions), we get a scar that reads TACTAG.
[[Image:screen-capture.png]]

For the afternoon, Olivia Ho-Shing and I finished assembly of the oligos of the suppressor tRNA codes. We assembled the document below:

http://gcat.davidson.edu/GcatWiki/images/5/5e/TRNAoligostoOrder.doc

We were assigned tasks of creating "cartoons" to illustrate the phenomenon that had occurred. No one could successfully do this however, because we could not understand how the piece had formed. The parts registry website provided wrong information on the restriction site of Spe1 which slowed our efficiency. This project turned into an effort to try to find two other different restriction enzymes that have complementary sticky ends other than Xba1 and Spe1. We want to get rid oc the scar that contains TACTAG.

We ended up deciding that a hybrid of two ideas was needed: restriction enzymes would need to be used to make several reporter gene sequences with different beginnings dependending on the suppressor tRNA sequence needed.

==Friday, June 5, 2009==

Our goal for this morning was to discuss the hybrid PCR approach to assemble the part that comes before the gene we wanted to express. We decided that we would have a BioBrick prefix and a 5mer followed by a sequence of nucleotides whose identity was up to us. Using PCR we would get the second double strand if we have the following:

BioBrick Prefix--ATG--5mer--gene until we get to restriction site

The PCR primer would attach to the ATG and the 5mer and would replicate the complementary base pairs to make a double stranded DNA sequence. As several of us were confused about PCR methods, we looked at the online link provided by Alyndria Thompson's link to the PCR interactive website which was very helpful. We also did some research to discover that the restriction enzyme BseRI (which is contained in the YFP that we wanted to alter) is not compatible with any other restriction enzyme. This is good because we did not want to plasmid containing this gene to close up with Xba1 once we digested it.

The team experienced some confusion with the hybrid idea and spoke to Clif Davis of Missouri Western University on the phone. He told us that there will be one part that is a fragment of the beginning of some nucleotides as a restriction cleaving site, ATG, the logical clause, and the start of the YFP gene that will be placed into a plasmid containing a space. To the left of the space will be the BioBrick Prefix and the right of the space will contain the rest of the genes in the YFP. Therefore, we decided not to alter any BioBrick ends and just look at restriction sites at the beginning of reporter proteins. The small fragment that will be placed into the plasmid will be made by PCR. The PCR will attach a primer onto the ATG and 5mer and replicate.

While we were discussing this, we expanded upon the idea that we can do whatever we want with the "suffix" portion between the 5mer and the beginning of YFP. We decided it would be best to remove ATG and add the first 5 amino acids and the restriction enzyme site. Along with this, if we have the 5mer coding for a certain amino acid (ie methionine) we could replace another codon that codes for methionine WITH that 5mer so long as this codon is contained within the first 15 nt. This way, we would not be altering the protein.

Olivia Ho-Shing came up with an idea of cascading PCR effect for longer logical clauses. Post translational modification of the protein was another one of her ideas. This applies because although we may code for the protein in a given way, the protein may modify or fix itself up to perform a slightly different function in its tertiary form.

Dr. Campbell suggested that we can look up restriction enzymes 400-500 base pairs into the reporter protein we would like to use; however, we have more flexibility in our procedure if we choose more "hardy" cutters. So instead, we could cut with EcoRI instead of with Xba1.

During the afternoon, we set about three tasks:
1) Which primers to use
2) Which promoters to use
3) Which restriction enzymes and how they will determine use of reporter proteins

Olivia Ho-Shing and I began looking up which restriction enzymes were strong so we could base our reporters on that. We began with Tet and RFP since those were the two reporters we had started out with choosing. I found a website that provided enzyme cleavage efficiency based upon the proximity of the site to the end of the PCR fragment that contains it. We discovered the following based upon the attached documents:

http://gcat.davidson.edu/GcatWiki/images/8/8f/Restriction_Enzyme_Cleavage_Efficiency.doc
http://gcat.davidson.edu/GcatWiki/images/9/9a/07Jun09_RE_Cutting_Efficiency_oh.doc

We decided that these two reporters contained efficient enzymes in a good quantity to choose from. On Monday, we will look through these enzymes once more and finalize which enzymes we would like to use with RFP and Tet.

==Sunday, June 7, 2009==

We did preparations for the mini prep this evening. The following notes are courtesy of Olivia Ho-Shing.

1. Sterically transfer ~2mL LB+AMP (AMP are ampicillin resistant plasmids) *Never put fluid back into bottle once it has entered pipette*
2. Use forceps to grab toothpick, scrap some frozen cells, rub in tube with top

'''Tubes'''

S03511 (2)

K091111

B0030

I715039

K091112

I715039

J31007 (2)

B0034

E1010

S03710

==Monday, June 8, 2009==

We reviewed the PCR process this morning as well as the restriction enzyme cleavage process.

Then, Leland Taylor, Olivia Ho-Shing and I all worked on converting the tRNA oligos we made to the template that Missouri Western used. The oligos we had previously made were different from the ones that Dr. Anderson had originally emailed us because further communication between Dr. Anderson and Missouri Western resulted in a better template.

We tried several times to minimize the number of oligos while keeping their individual lengths under 80bp. We managed to do this in the end by creating 7 oligos, 2 of which are variable between the suppressor tRNAs.
We reviewed the following document and finalized the oligos that we needed to order for the frameshift suppressor tRNAs. We decided to keep both the 10bp and 9bp anticodons of CCAUC.

I also looked up the amino acids that corresponded to the FS (frameshift suppressor) tRNAs for future use:

1. CUAGU = Leu
2. CCCUC = Pro
3. CGGUC = Arg
4. CCACU = Pro
5. CCAUC* = Pro
*9 and 10 bp anticodons

Also, we have decided to use NcoI as the restriction enzyme for RFP. This cleavage site occurs 419 bp into the gene. We have decided to use BamHI as the enzyme for Tetracycline Resistance and the cleavage site occurs 290bp into the gene. Both of these enzymes are very powerful and hardy.

We also looked over Missouri Western's tRNA oligos and primer oligos which we approved.
Our oligos were also approved by Missouri Western and Dr. Campbell ordered them while we supervised.

In the afternoon we did the miniprep. The mini prep directions were as appear in the link below.

http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_MiniPrep.html

We then worked with the Nano Prep to dilute our solution of DNA to an absorption of 1.5 and then we made labels for the tubes. The chart below shows relevant information we obtained from the NanoDrop for each of the reporter plasmids.

[[Image: Screen-capture-6.png]]

==Tuesday, June 9, 2009==

'''Preparations for restriction enzymes digestion of plasmids'''

Purpose: To determine the size of the plasmid inserts and ensure that plasmids are what we extracted from the cells during the mini-prep.

Things we need:
DNA ~3uL
Buffer 10x (Want to dilute to 1x) so add 2uL
Enzyme 1 ~1uL
Enzme 2 ~1uL
Water ~13uL
Final Volunter about 20 uL.

Need to choose the right buffer for the restriction enzymes. All buffers come in 10x (10 times more concentrated than what you want it to be when you use it). [http://gcat.davidson.edu/GcatWiki/index.php/Davidson_Missouri_W/Double_Digest_Guide Buffer Website]

Total volume of enzymes has to be 10% or less than final volume (most volume 2uL). Barely submerge the pipet tip into the solution when pipeting the enzymes.
Star (*) activity: Restriction enzyme stops cutting specifically and starts cutting everywhere.

DNA can't be seen by itself so we add EtBr (the bigger the insert, the more will bind) so bigger strands appear brighter. samples.

The order of entry doesn't matter, but make sure to enter the enzymes last.

Will have lots of labeled tubes and will put 3uL of appropriate plasmids into the tubes.
In the meantime, we'll make a "Master Mix" of all the other ingredients in a bigger tube. Always make enough volume for one extra tube.

Enzymes usually have an optimal temperature of 37C. We will let this reaction go for an hour and then we will run it on a gel.

'''Gel Electrophoresis Protocol'''

Website online for ideal gel concentration for resolving different sizes of molecules [http://gcat.davidson.edu/iGEM08/gelwebsite/gelwebsite.html Gel Concentration Website].
See protocol on the Davidson/Missouri Western Wookie for running molecules in the gel and how to digest with restriction enzymes.

'''What's Next?'''

1. 5' end additions
a. Using PCR
b. Cut at the restriction sites EcoR1 and either Bam H1 or Nco1
c. Cut plasmid and reporter with same restriction enzymes as above
d. Run a gel and purify "keeper" DNA (Ligate transform, screen, sequence)
e. Then, we take the "keeper" DNA and combine it with the insert from part

We can have steps d and e ready in advance.

2. tRNAs
a. Assemble the oligos into genes (Requires lots of calculations beforehand, but the actual steps )
b. Plasmids cut up with EcoR1 and Pst1 '''WE CAN USE THE "WASTE" PLASMID FROM THE RESTRICTION ENZYME DIGEST'''
c. Gel purify the plasmids (Ligate transform, screen, sequence)

The following tables include Master Mix measurements, agarose gel concentrations and buffers.
http://gcat.davidson.edu/GcatWiki/images/0/08/Shamitapic3.png
http://gcat.davidson.edu/GcatWiki/images/d/d1/Shamitapic4.png
http://gcat.davidson.edu/GcatWiki/images/6/6f/Shamitapic5.png

We made the Master Mix with our DNA and put it in the incubator at 10:40 AM.

'''Preparing the Gels'''

Now, we will be making 2 gels, 1 at 0.8% and 2.5% (weight to volume percent). The gel volume is 60mL.

'''Definition of 1% solution:
1 gram of agarose/ 100mL buffer'''

So for a 0.8% solution: 0.8 grams agarose/100mL buffer AND we need 60mL (fixed volume for casts run) so multiply that by 0.6.
Final concentration:

.48 grams agarose/60mL buffer

1 uL of EtBr stock for 60mL of gel.
EtBr is in the buffer so it is constantly replenishing itself even though it is moving toward one end in order to make bands on the other end visible.

After Dr. Campbell's demonstration, we each helped prepare a 0.8% buffer solution using the agarose powder, buffer and EtBr. We mixed together the first two substances in a 200mL Erlenmeyer flask and heated it for 120 seconds in the microwave. We then added EtBr and poured the gel solution into the gel cast.

'''Agar Plate'''

After lunch, we grew some more cells for each of the 11 parts in an agar plate labeled "LBAMP-AMC-9 June" so that so would have extra cells that could be stored for up to 2 weeks.
We made small circles on the agar plate and numbered them 1-11. The numbers corresponded to the following parts. We put the plate in the incubator overnight to let the cells grow.

1. B0030 AMC
2. K091111
3. S03710
4. I715039 OEH
5. S03511 ORH
6. I715039 AST
7. B0030 SDP
8. K091112
9. J31007 LJH
10. J31007 REC
11. S03511

'''Unloading the Gels'''

The following shows which parts we entered in which gels:

[[Image: screen-capture-2.png]]

We unloaded the 2.5% agarose first and after observing the bands under a UV light, put the gel into the BioRad machine to observe the bands. The image below is of a Molecular Weight Marker that shows the number of bp in control fragments of DNA according to the distance they travel down the gel.

[[Image: DNA_molecular_weight_marker_gel.png]]

We obtained the following printout for the first gel that was 2.5% Agarose.
[[Image: 2009-06-09_14hr_44min.jpg]]

The bands in the first well at the bottom are those of the molecular weight marker. We skipped the lane above (to the right) of the MWM and entered the rest of the inserts as described in the above table. A light band is visible for each of the 4 other wells. In Lanes 3 and 4 we concluded we are supposed to have approximately 45 bp because the insert is 15bp + 30 bp on either side of the insert (Xba1 and Spe1 restrictions sites + sticky ends + extra bases). We used the same logic for Lanes 5 and 6 which are supposed to contain approximately 86 base pairs.

One discrepancy in this gel, however, is that Lanes 5 and 6, which according to their labels respectively contain the LacIQ promoter and the Lac IQ1 promoter. According to the information online at the parts registry website, pLacIQ1 should be the same length as pLacIQ. The gels do not show this, but instead show that the sample in Lane 5 is smaller than that in Lane 6 because Lane 5 traveled further. Leland Taylor and Romina Clemente sent out an email to Pallavi Penumetcha to ask about this because she had written a paper about this.

We then unloaded the 0.8% agarose gel and then placed this in the BioRad machine under the same protocol as above. The following image is the 0.8% Agarose.

[[Image:2009-06-09_15hr_21min.jpg]]

We observed that the bands in Lane 1 (which is now at the top) are examples of high intensity bands. The plasmids contained in the original well are twice the intensity of the insert.

In Lane 5, however, we observed several different bands. This could be due to the extra DNA that was sitting on the top of the well. We decided to re-expose the gel to get better observations on Lanes 3 and 4 because the bands were not as intense. We anticipated seeing a band at about 250 bp because this was the size of the insert (plus extra bp). After re-exposing the gel, we did not find any difference and still did not see any bands in these lanes. We have decided to redo the gel on the part S03511 and so we prepared the master mix with that DNA and incubated it so that we could run the gel tomorrow.

We also cut out the "empty" plasmids from the gel so that we could use this as vectors for the coding sequence of the suppressor tRNAs.

Tomorrow we will be planning out specific tasks for people once the oligos arrive.

==Wednesday, June 10, 2009==

We began this morning by making the agarose gel with the same protocol as yesterday. This time, since we are just re-doing both S03511 inserts, we will have a 1.8% agarose gel.

'''OUTLINE: WHERE DO WE STAND?'''

Yesterday
a. Isolated empty plasmids
b. Verified most inserts
c. Waiting for:
>tRNA oligos
>Primers
a. Will amplify the reporter gene in the plasmid itself

Next?
a. When Primers Arrive
> Amplify the reporter genes with primers (PCR takes ~3 hours)
> Calculate how much reporter plasmid we will need (Start with 50x more than we actually will need)
> Cut amplified sequence (EcoR1 and BamH1/Nco1) then purify what we need
> Cut reporter plasmid (EcoR1 and BamH1/Nco1) then purify what we need
> Ligate amplified sequence and reporter plasmid
b. When tRNA oligos arrive
> Calculate how much of each oligo we will need (takes a while...)
> "Boil and cool" tRNA oligos together
> Ligate fragments (dsDNA) into plasmids we isolated yesterday (~5 minutes)
> Transform the plasmids (~20 minutes)

* LOOK THROUGH LIGATION PROTOCOL (How to assemble genes)
* LOOK THROUGH TRANSFORMATION PROTOCOL

''What we need to get done today'':
1. Prepare the control for the Tet Resistance by BioBricking the part pLac+RBS (S03511) with the TetA (J31007).
2. Check the part number for RFP and also for pLac+RBS+RFP
3. Read through ligation protocol
4. See how many nanograms of plasmid we will need for the tRNAs and for the inserts and multiply this number by 50.
> Check if we have enough plasmids from our mini-prep
5. Purify the plasmids from the gel

After we made this outline, Leland, Olivia and I analyzed the gel we ran this morning and did not see a more than one band in the 2 lanes that contained DNA from S03511 (image below). Since we did not see multiple bands in yesterday's gel either, we conclude that there is something wrong with the DNA rather than with the restriction enzymes that we used to cut it. We concluded that there is probably not an insert in this plasmid both the part and the plasmid are useless.

[[Image: 2009-06-10 10hr 30min.jpg]]

Now, we do not have an immediately accessible promoter. We have decided to order the pBAD promoter from Missouri Western and we will attach the RBS+Tet to this plasmid. Our insert for this will be the larger gene because it is easier to manipulate in the gel than the small gene.

Alyndria Thompson and Romina Clemente are figuring out the size of the plasmids including the reporter genes.

After lunch we performed several steps to isolate and purify the gel to get the empty plasmids will be the vectors for the tRNA DNA. Then, we used the NanoDrop to assess the purity of our DNA. We found that the plasmids were more pure than we had expected. The following values were obtained:

Abs 1.850
A-260 10 mm path 0.172
A-280 10 mm path 0.075
260/280 2.28
260/230 0.09

We then followed the protocol for preparing the agar plates. We put the plate with the agar solution in the Autoclave.

While the solution was in the autoclave, we discussed that we don't really need a promoter in front of RBS+Tet for the gene to be expressed because Tet expression is so strong that the gene would translate without a promoter. However, it would be a good idea to have the promoter because if it was possible to reinforce the translation of the reporter protein, we wanted to do that.

We found that there were some tubes in the lab fridge that contained the pBAD and pLac promoters with RBS. We decided to use these as the promoter and RBS for our experiments. The pLac promoter will be used for RFP and the pBad promoter used with Tet Resistance. We decided to use the pBad with Tet because it is weaker in comparison to pLac. As expression of the Tet gene can be toxic to a cell, we do not want to overexpress the gene with a stronger promoter.

Once the agar solution was sterilized in the Autoclave, we removed it, cooled it, and pipetted the antibiotic resistance into it. We then poured the solution into the plates, removed any bubbles, and let the gel form.

Alyndria Thompson and Romina Clemente concluded that we had more than enough recieving vectors for both the RFP and Tetracycline genes. Since we need 50 nanograms, it is wise to have at least 50 times that much that we can work with, so we need at least 2,500 ng.

RFP 1: 77.6 x 4 = 310.4 ng/uL
x 20 uL = 3,272 ng
RFP 2: 59.5 x 4 = 238 ng/uL
x 20 uL = 4,760 ng
Tet: 40.9 x 4 = 163.6 ng/uL
x 20 uL = 3,272 ng

According to these calculations we have more than 50 times what we need for both instances of RFP and Tet.

Tomorrow, we can prepare the Master Mix for the digestions of both reporter plamids and then add the restriction enzymes once they arrive. Then, we can follow through with the rest of the plans we had made above in the section titles "Next?".

==Thursday, June 11, 2009==

We began this morning by calculating how many uL we would need for each of the mixes for digesting our reporter plasmids. We decided to double the final volume of each mix to 40uL instead of 20uL. Since we knew the concentration of each plasmid and also knew we needed 2500 ng, we cross multiplied to find the uL of DNA we would need for each.

Concentration Calculations:
310.4 ng/1 uL = 2500ng/x uL
x = 8.05 uL

Here is a chart indicating how much of each we used for the 2 mixes.

[[Image: DoubledigestRFPTetR.png]]

On the Promega website, we found a list of compatible buffers for double digestions. Buffers E and H are compatible for the Tet plasmid and RFP plasmid digestions respectively. We fortunately have both buffers in stock.

[[Image:BamEcoR1.png]] [[Image:NcoEcoR1.png]]

We waited for our waited for the restriction enzymes to arrive and in the meantime, we transformed the plasmids with pLac+RBS, pBad, and pBad+RBS into ''E.coli'' cells. We want the ''E.coli'' cells to replicate the plasmids with these genes because we have a limited amount of genes. [http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_Transformation.html Zippy Plasmid Transformation]

Once our oligos and restriction enzymes arrived, we got to work right away!

'''PCR (Polymerase Chain Reaction)'''

We calculated how much plasmid we will need for the PCR. We decided to dilute the concentration of our plasmids a hundred fold so that we could get pipetable amounts.

''Change in Concentrations''
310.4 ng/uL x 1/100 (dilution factor) = 3.104 ng/uL (RFP)
163.6 ng/uL x 1/100 (dilution factor) = 0.164 ng/uL (Tet)

''Example shown with RFP''
3.104 ng/1 uL = 1ng/ x uL, x = .32
For Tet, x = .61

We also calculated how much of each primer we would need with the following equation:

C1V1 = C2V2
(100 uM) (x) = (1 uM)( 100 uL)
x = 1 uL

[[Image: screen-capture-8.png]]

Each of the 5 of us made had 2 tubes (one for each reporter protein) because there were 5 different forward primers.To each tube, we added the template DNA (either RFP or Tet), water, the respective reverse primer (either RFP or Tet), and the forward primer depending on which 5mer we had chosen. My 5mer was CCAUC. Finally, we added the Master Mix last because this contained the enzyme (DNA polymerase) that would amplify our desired sequence.

[[Image: Pcrtubes.png]]

I had

We followed the temperature cycles so that PCR could occur. The annealing temperature needed to be 5C less than the smallest melting temperature of the primers. Since the reverse primer of RFP had a melting temp of 66C, we set the temperature of the 3rd cycle on 61C.

[[Image: Temperaturecycle.png]]

'''tRNA Oligo Assembly'''

We needed to calculate the concentration of each of the oligo before carrying out the assembly. The stock concentration of each of the oligo was 100 uM. Since we wanted to have 5uM of the oligo in the test tube, and we know that final volume of the test tube is 20 uL, we just cross multiplied to find the amount we needed to add to the tubes to get this concentration.

(100uM)x = (5uM)(20uL)
x = 1 uL

We added 1 uL of each oligo to the tubes.

We had six total tRNAs we needed to assemble so each of us took one FML (one person took two) and to each tube we added 1uL of each oligo for a total of 7uL, 2uL of buffer, and 11uL of water for a total volume of 20 uL. Five of the oligos were constant and 2 varied for each of the different tRNAs. So everyone pipetted the same 5 oligos and everyone also had 2 unique oligos to add. When all of the oligos were added and the tubes ready, we boiled the tubes at 50C for 5 minutes. We then allowed the tubes to remain in the water to cool very slowly overnight so that the annealing process could take place.

The last thing we did at the end of the day was finish off our plasmid digestion that we had begun in the morning. We added the buffer, DNA, water, and restriction enzymes and placed this in the incubator for overnight digestion.

'''To Do''':

1. Isolate our desired dsDNA from the PCR.
2. Verify our tRNA inserts and eventually transform these into plasmids.
3. Check our agar plates to see colonies that have replicated our pLac+RBS, pBAD+RBS, and pBAD genes.

==Friday, June 12, 2009==

This morning, we began by outlining the main items on today's agenda.

1. tRNAs: Ligation, Transformation
2. Digestion reporters (run all of it and gel purify)
3. Transformed cells
4. PCR: Gel run an aliquot to verify (5uL)

We used the equation given by the Ligation protocol to calculate how many nanograms of insert we would need. Initially, we started out calculated the MW average of the 9 and 10 anticodon loops of tRNAs which were 15828.23 ug/umols and 159016.6 ug/umols respectively. We then saw, however, that these would not be helpful values in the process. Instead we started out with the equation below to find the final ng of insert we would need for the ligation.

[[Image: ligationcalculations.png]]

We calculated our initial concentration in ng/uL of insert as shown below.

[[Image: ligationequation.png]]
X nanograms of insert = [(2)(144bp)(50 ng linearalized plasmid)]/2038bp
X = 7.0657 ng of insert

We wanted to find the amount of uL of insert we would need to use. We diluted the initial concentration 50x because we wanted a pipettable amount of insert. Since we knew we wanted the final amount of nanograms to be 7.0657, we calculated as shown below:

''DILUTION'': 555.48/50 = 11.012
''CALCULATION'': 11.012 ng/ 1uL = 7.065 ng/ x uL
x = .64 uL of insert

After we had done these calculations, half of the group started to carryout the ligation of the tRNA inserts into empty plasmids. We needed 7 ligations total: 6 were the tRNAs we needed, while the last one is a negative control. These are the final amounts of what we used:

[[Image: Trnaligation.png]]

The other half of the group (which included me) prepared the agarose gels so that we could run gels for the digested reporters and the PCR inserts. We calculated that the digested reporters needed an agarose of 4% and the PCR reporters needed an agarose of 1.45%. We allowed this gel to form for about 45 minutes to an hour. We prepared both the gels and allowed them to run through lunch.
After lunch, we took pictures of our gels and noticed that the third lane in the PCR gel (third lane from the top in the bottom photo) did not develop a band. The lane was supposed to contain the CUAGU insert for Tetracycline Resistance.

[[Image: 2009-06-12 15hr 54min.jpg]] [[Image:2009-06-12_16hr_07min.jpg]]

Romina Clemente and I decided to repeat this PCR using the TetResistance and RBS+TetResistance plasmids as templates. We labeled J31007 as "2*" and S03710 as "2!". We had to recalculate how much template DNA to use. We let the PCR run overnight. We also verified that the digestion fragments (both the plasmid and the insert) on the other gel were the correct size. The plasmids, which are the brighter fragment in the gel with only 2 lanes, contain the plasmid base pairs along with the rest of our reporter genes. We isolated these plasmids from the gel today and stored them for purification on Monday.

In the meantime, other members of our lab had finished the ligation of the tRNA genes to the empty plasmids and were now transforming these into competent cells. They placed the agar plate with these cells in the fridge overnight.

We also observed the transformations that we had performed yesterday and noticed that I13453 did not form any colonies. J04500 and J3102 did form colonies.

To Do:
1. PCR: Dig, Ligate, Transform
Clean and concentration column, digest, clean and concentration column, ligate.
2. Gel purification of our digestion plasmids
3. Check if colonies have grown for our tRNAs

==Saturday, June 13, 2009==

Alyndria Thompson came in on Saturday morning to remove the tRNA plates and put the PCR reactions in the fridge.

'''tRNA Colonies'''

She counted the colonies of tRNA on each plate.

[[Image:TRNA_colonies_6_13_09.png]]

She placed the plates in the large fridge on the top shelf at 9:23 am. She talked to Dr. Campbell who said we will have to try to "work them up".

'''PCR'''

Products were placed in orange rack of smaller fridge. [2*, 2!]

==Sunday, June 14, 2009==

'''Second Ligation and Transformation of tRNA inserts'''

We decided to re-ligate and re-transform all of the tRNA inserts because we did not get very good yields of colonies from the transformations.
We are using the same measurements for this ligation as we had used on Friday.

[[Image: Trnaligation.png]]

After ligation, we transformed the cells. Using the following measurements, we transferred the competent cells into new tubes.

40uL of competent cells
10uL of ligation mixture
60uL of LB media

After spreading the mixture onto the agar plates, we incubated the plates at 10:56 pm.

We also stored liquid cultures of the colonies that greater than a 2:1 variable:negative control ratio just in case we wanted to use them for mini preps in the future. These included #1 (3 samples), #4 (2 samples), #5 (1 sample), and #6 (2 samples).

'''To Do''':
1. Count colonies obtained from second transformation
2. Clean PCRs: Dig, ligate, transform
3. Run gel for 2 Tet PCRs, clean
4. Gel purify receiving vector

IGEM Notebook

2009-06-15T04:05:12Z

ShPunjabi: /* Sunday, June 14, 2009 */

==Wednesday, June 3, 2009==

Romina Clemente and I are trying to find suitable reporter proteins to use. Yesterday, Leland Taylor and Alyndria Thompson were working on ways to insert the gene sequences into the plasmid. Upon seeing how they wanted to manipulated the reporter gene to include the logical clauses, we came up with a few criteria for the reporter genes we would use. The following criteria for genes are listed in order of the broadest aspect to look at to the narrowest aspect:
a) Doesn't contain restriction sites for the 4 restriction enzymes (EcoR1, Xbal, Spel, Pst1) used to cleave the Biobrick part out of the plasmid.
b) Contains 6 cutter restriction sites.
c) These restriction enzymes aren't blunt (cleave straight down at one spot).
d) These restriction sites are close to thge 5' (beginning) end of the sequence.
e) These enzymes are easiest to work with and cheapest.
We are finding the part numbers of the reporter genes we want to use (antibiotic resistance, fluorescence, LacZ) through our own GCAT because we know these ones work. We are then locating these parts on the parts registry [1] website. We copied and pasted the gene sequences we obtained from the registry onto the ApE software [2]. From here, we were able to generate a genetic map of each gene that outlined each restriction site that fit our criteria. We put each genetic map, alongside the part number used, into a Word document.

http://gcat.davidson.edu/GcatWiki/images/0/0e/Restriction_Site_Mapping_on_Reporter_Genes.doc

Later on in the day, we decided that the best way to test for suppression would involve placing the 5mers at the beginning of the reporter gene instead of inserting them into the reporter gene. To do this, we decided that the gene along with its start codon need to be expressed after the 5mer. We will insert a BioBrick plasmid with a 5mer and start codon that will be incorporated before the gene:
RBS-6-8nt-ATG-5mer--ATG-gene

Leland Taylor assembled the oligos that we will need for the BioBrick pieces.

In order to test whether the reporter protein would be expressed regardless of the 5mer being suppressed or not, we would need to remove the suppressor tRNAs from the cell.

Meanwhile, I helped figure out the coding sequence that we will need for the suppressor tRNAs that will bind to the 5mers. We decided that we would be using the following suppressor tRNAs on the Davidson side: CUAGU, CCCUC, CGGUC, CCAUC, and CCACU. We searched through several papers, with little result and finally emailed Dr. Christopher Anderson of UC Berkeley to request the DNA sequence. He emailed us back with a generic DNA code that we could use to create the tRNA; however, for each suppressor tRNA we would use, we would need to change base pairs in the anticodon loop.

The length of the tRNA is 92 nt. The anticodon loop is comprised of 9 nt. Once we have BioBricked the DNA sequence with 4 restriction sites and supplemental nucleotides, the length of the entire gene is 144 nucleotides. This is made up of the 92 nt +22 nt on each side of the sequence (44) + 4 nt on each side of the restriction ends (8).

At the end of the day, we decided to use tetracycline resistance and RFP as our reporter proteins for this tester experiment.

==Thursday, June 4, 2009==

This morning, I began the day by learning how to use the PEARL prgram. Olivia Ho-Shing sat with me and walked me through the program script. And then, Leland Taylor and Shashank Suresh had a problem they wanted us to solve. Leland explained that we need to make sure there is a stop codon in the gene if we fail to suppress the 5mer. The stop codon needs to be before we find another 5mer. They also wanted us to run through both the RFP and Tet Resistance genes to make sure that they did not contain any of the anticodon sequences from our 5 frameshift suppressor tRNAs so as to throw the translation out of frame. They further wanted us to confirm that if we do have successful suppression, there are no stop codons in the middle of the RFP and Tet Resistance genes. PEARL did not find any matches for the suppressor tRNAs within genes. We found 3 matches for stop codons UAA. Two of these occured at the end of the Tet and one at the end of RFP, where they should normally be to end translation into a protein.

Following this, Olivia Ho-Shing and I tried to determine how we can add BioBrick ends to our single stranded DNA sequences that code for the suppressor tRNAs. Our ultimate goal is to find complements for these single strands so they can be put into plasmids. We found the standard prefixes and suffixes for sequences that do not contain ATG (becuase this sequence is a functional RNA and will not be translated) and we placed these before and after the altered 92bp sequence that Dr. Anderson provided us with. The prefixes and suffixes are necessary because they are the extra nucleotides that will be mimicking a "restriction enzyme cutting site". The single strand appeared as below after the Bio Brick prefixes and suffixes were added:

22ntPREFIX--32nt--VARIABLE ANTICODON LOOP--51nt--21ntSUFFIX

We used the suppressor codon CUAGU as our example and placed its appropriate anticodon loop in the sequence. We put this into the lancelator that would give us the other strand as well as the oligos we would need for ideal construction of this double stranded DNA fragment to put into the plasmid. We recieved a total of 4 oligos, 2 of which were variable and 2 of which were constant for all 6 different suppressor tRNAs. Using this double strand, we simulated a restriction enzyme digestion by EcoRI and PstI. We took off the G at both ends of the top strand and then removed the first five nucleotides on each end of the bottom strand. This made the top strand longer than the bottom one. We then proceeded to take the two variable strands and changed the anticodon loop nt for each suppressor tRNA we used.

Around lunchtime, we encountered a problem with the BioBrick scar in the "ATG-5mer subpiece" of the test project. It turns out that after annealing the sticky ends of the Xba1 and Spe1 sites, the scar created had a stop codon TAG (UAG) that was in frame with the suppressed codon. When the Xba1 site on insert anneals to the Spe1 site on the plasmid (the green portions), we get a scar that reads TACTAG.
[[Image:screen-capture.png]]

For the afternoon, Olivia Ho-Shing and I finished assembly of the oligos of the suppressor tRNA codes. We assembled the document below:

http://gcat.davidson.edu/GcatWiki/images/5/5e/TRNAoligostoOrder.doc

We were assigned tasks of creating "cartoons" to illustrate the phenomenon that had occurred. No one could successfully do this however, because we could not understand how the piece had formed. The parts registry website provided wrong information on the restriction site of Spe1 which slowed our efficiency. This project turned into an effort to try to find two other different restriction enzymes that have complementary sticky ends other than Xba1 and Spe1. We want to get rid oc the scar that contains TACTAG.

We ended up deciding that a hybrid of two ideas was needed: restriction enzymes would need to be used to make several reporter gene sequences with different beginnings dependending on the suppressor tRNA sequence needed.

==Friday, June 5, 2009==

Our goal for this morning was to discuss the hybrid PCR approach to assemble the part that comes before the gene we wanted to express. We decided that we would have a BioBrick prefix and a 5mer followed by a sequence of nucleotides whose identity was up to us. Using PCR we would get the second double strand if we have the following:

BioBrick Prefix--ATG--5mer--gene until we get to restriction site

The PCR primer would attach to the ATG and the 5mer and would replicate the complementary base pairs to make a double stranded DNA sequence. As several of us were confused about PCR methods, we looked at the online link provided by Alyndria Thompson's link to the PCR interactive website which was very helpful. We also did some research to discover that the restriction enzyme BseRI (which is contained in the YFP that we wanted to alter) is not compatible with any other restriction enzyme. This is good because we did not want to plasmid containing this gene to close up with Xba1 once we digested it.

The team experienced some confusion with the hybrid idea and spoke to Clif Davis of Missouri Western University on the phone. He told us that there will be one part that is a fragment of the beginning of some nucleotides as a restriction cleaving site, ATG, the logical clause, and the start of the YFP gene that will be placed into a plasmid containing a space. To the left of the space will be the BioBrick Prefix and the right of the space will contain the rest of the genes in the YFP. Therefore, we decided not to alter any BioBrick ends and just look at restriction sites at the beginning of reporter proteins. The small fragment that will be placed into the plasmid will be made by PCR. The PCR will attach a primer onto the ATG and 5mer and replicate.

While we were discussing this, we expanded upon the idea that we can do whatever we want with the "suffix" portion between the 5mer and the beginning of YFP. We decided it would be best to remove ATG and add the first 5 amino acids and the restriction enzyme site. Along with this, if we have the 5mer coding for a certain amino acid (ie methionine) we could replace another codon that codes for methionine WITH that 5mer so long as this codon is contained within the first 15 nt. This way, we would not be altering the protein.

Olivia Ho-Shing came up with an idea of cascading PCR effect for longer logical clauses. Post translational modification of the protein was another one of her ideas. This applies because although we may code for the protein in a given way, the protein may modify or fix itself up to perform a slightly different function in its tertiary form.

Dr. Campbell suggested that we can look up restriction enzymes 400-500 base pairs into the reporter protein we would like to use; however, we have more flexibility in our procedure if we choose more "hardy" cutters. So instead, we could cut with EcoRI instead of with Xba1.

During the afternoon, we set about three tasks:
1) Which primers to use
2) Which promoters to use
3) Which restriction enzymes and how they will determine use of reporter proteins

Olivia Ho-Shing and I began looking up which restriction enzymes were strong so we could base our reporters on that. We began with Tet and RFP since those were the two reporters we had started out with choosing. I found a website that provided enzyme cleavage efficiency based upon the proximity of the site to the end of the PCR fragment that contains it. We discovered the following based upon the attached documents:

http://gcat.davidson.edu/GcatWiki/images/8/8f/Restriction_Enzyme_Cleavage_Efficiency.doc
http://gcat.davidson.edu/GcatWiki/images/9/9a/07Jun09_RE_Cutting_Efficiency_oh.doc

We decided that these two reporters contained efficient enzymes in a good quantity to choose from. On Monday, we will look through these enzymes once more and finalize which enzymes we would like to use with RFP and Tet.

==Sunday, June 7, 2009==

We did preparations for the mini prep this evening. The following notes are courtesy of Olivia Ho-Shing.

1. Sterically transfer ~2mL LB+AMP (AMP are ampicillin resistant plasmids) *Never put fluid back into bottle once it has entered pipette*
2. Use forceps to grab toothpick, scrap some frozen cells, rub in tube with top

'''Tubes'''

S03511 (2)

K091111

B0030

I715039

K091112

I715039

J31007 (2)

B0034

E1010

S03710

==Monday, June 8, 2009==

We reviewed the PCR process this morning as well as the restriction enzyme cleavage process.

Then, Leland Taylor, Olivia Ho-Shing and I all worked on converting the tRNA oligos we made to the template that Missouri Western used. The oligos we had previously made were different from the ones that Dr. Anderson had originally emailed us because further communication between Dr. Anderson and Missouri Western resulted in a better template.

We tried several times to minimize the number of oligos while keeping their individual lengths under 80bp. We managed to do this in the end by creating 7 oligos, 2 of which are variable between the suppressor tRNAs.
We reviewed the following document and finalized the oligos that we needed to order for the frameshift suppressor tRNAs. We decided to keep both the 10bp and 9bp anticodons of CCAUC.

I also looked up the amino acids that corresponded to the FS (frameshift suppressor) tRNAs for future use:

1. CUAGU = Leu
2. CCCUC = Pro
3. CGGUC = Arg
4. CCACU = Pro
5. CCAUC* = Pro
*9 and 10 bp anticodons

Also, we have decided to use NcoI as the restriction enzyme for RFP. This cleavage site occurs 419 bp into the gene. We have decided to use BamHI as the enzyme for Tetracycline Resistance and the cleavage site occurs 290bp into the gene. Both of these enzymes are very powerful and hardy.

We also looked over Missouri Western's tRNA oligos and primer oligos which we approved.
Our oligos were also approved by Missouri Western and Dr. Campbell ordered them while we supervised.

In the afternoon we did the miniprep. The mini prep directions were as appear in the link below.

http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_MiniPrep.html

We then worked with the Nano Prep to dilute our solution of DNA to an absorption of 1.5 and then we made labels for the tubes. The chart below shows relevant information we obtained from the NanoDrop for each of the reporter plasmids.

[[Image: Screen-capture-6.png]]

==Tuesday, June 9, 2009==

'''Preparations for restriction enzymes digestion of plasmids'''

Purpose: To determine the size of the plasmid inserts and ensure that plasmids are what we extracted from the cells during the mini-prep.

Things we need:
DNA ~3uL
Buffer 10x (Want to dilute to 1x) so add 2uL
Enzyme 1 ~1uL
Enzme 2 ~1uL
Water ~13uL
Final Volunter about 20 uL.

Need to choose the right buffer for the restriction enzymes. All buffers come in 10x (10 times more concentrated than what you want it to be when you use it). [http://gcat.davidson.edu/GcatWiki/index.php/Davidson_Missouri_W/Double_Digest_Guide Buffer Website]

Total volume of enzymes has to be 10% or less than final volume (most volume 2uL). Barely submerge the pipet tip into the solution when pipeting the enzymes.
Star (*) activity: Restriction enzyme stops cutting specifically and starts cutting everywhere.

DNA can't be seen by itself so we add EtBr (the bigger the insert, the more will bind) so bigger strands appear brighter. samples.

The order of entry doesn't matter, but make sure to enter the enzymes last.

Will have lots of labeled tubes and will put 3uL of appropriate plasmids into the tubes.
In the meantime, we'll make a "Master Mix" of all the other ingredients in a bigger tube. Always make enough volume for one extra tube.

Enzymes usually have an optimal temperature of 37C. We will let this reaction go for an hour and then we will run it on a gel.

'''Gel Electrophoresis Protocol'''

Website online for ideal gel concentration for resolving different sizes of molecules [http://gcat.davidson.edu/iGEM08/gelwebsite/gelwebsite.html Gel Concentration Website].
See protocol on the Davidson/Missouri Western Wookie for running molecules in the gel and how to digest with restriction enzymes.

'''What's Next?'''

1. 5' end additions
a. Using PCR
b. Cut at the restriction sites EcoR1 and either Bam H1 or Nco1
c. Cut plasmid and reporter with same restriction enzymes as above
d. Run a gel and purify "keeper" DNA (Ligate transform, screen, sequence)
e. Then, we take the "keeper" DNA and combine it with the insert from part

We can have steps d and e ready in advance.

2. tRNAs
a. Assemble the oligos into genes (Requires lots of calculations beforehand, but the actual steps )
b. Plasmids cut up with EcoR1 and Pst1 '''WE CAN USE THE "WASTE" PLASMID FROM THE RESTRICTION ENZYME DIGEST'''
c. Gel purify the plasmids (Ligate transform, screen, sequence)

The following tables include Master Mix measurements, agarose gel concentrations and buffers.
http://gcat.davidson.edu/GcatWiki/images/0/08/Shamitapic3.png
http://gcat.davidson.edu/GcatWiki/images/d/d1/Shamitapic4.png
http://gcat.davidson.edu/GcatWiki/images/6/6f/Shamitapic5.png

We made the Master Mix with our DNA and put it in the incubator at 10:40 AM.

'''Preparing the Gels'''

Now, we will be making 2 gels, 1 at 0.8% and 2.5% (weight to volume percent). The gel volume is 60mL.

'''Definition of 1% solution:
1 gram of agarose/ 100mL buffer'''

So for a 0.8% solution: 0.8 grams agarose/100mL buffer AND we need 60mL (fixed volume for casts run) so multiply that by 0.6.
Final concentration:

.48 grams agarose/60mL buffer

1 uL of EtBr stock for 60mL of gel.
EtBr is in the buffer so it is constantly replenishing itself even though it is moving toward one end in order to make bands on the other end visible.

After Dr. Campbell's demonstration, we each helped prepare a 0.8% buffer solution using the agarose powder, buffer and EtBr. We mixed together the first two substances in a 200mL Erlenmeyer flask and heated it for 120 seconds in the microwave. We then added EtBr and poured the gel solution into the gel cast.

'''Agar Plate'''

After lunch, we grew some more cells for each of the 11 parts in an agar plate labeled "LBAMP-AMC-9 June" so that so would have extra cells that could be stored for up to 2 weeks.
We made small circles on the agar plate and numbered them 1-11. The numbers corresponded to the following parts. We put the plate in the incubator overnight to let the cells grow.

1. B0030 AMC
2. K091111
3. S03710
4. I715039 OEH
5. S03511 ORH
6. I715039 AST
7. B0030 SDP
8. K091112
9. J31007 LJH
10. J31007 REC
11. S03511

'''Unloading the Gels'''

The following shows which parts we entered in which gels:

[[Image: screen-capture-2.png]]

We unloaded the 2.5% agarose first and after observing the bands under a UV light, put the gel into the BioRad machine to observe the bands. The image below is of a Molecular Weight Marker that shows the number of bp in control fragments of DNA according to the distance they travel down the gel.

[[Image: DNA_molecular_weight_marker_gel.png]]

We obtained the following printout for the first gel that was 2.5% Agarose.
[[Image: 2009-06-09_14hr_44min.jpg]]

The bands in the first well at the bottom are those of the molecular weight marker. We skipped the lane above (to the right) of the MWM and entered the rest of the inserts as described in the above table. A light band is visible for each of the 4 other wells. In Lanes 3 and 4 we concluded we are supposed to have approximately 45 bp because the insert is 15bp + 30 bp on either side of the insert (Xba1 and Spe1 restrictions sites + sticky ends + extra bases). We used the same logic for Lanes 5 and 6 which are supposed to contain approximately 86 base pairs.

One discrepancy in this gel, however, is that Lanes 5 and 6, which according to their labels respectively contain the LacIQ promoter and the Lac IQ1 promoter. According to the information online at the parts registry website, pLacIQ1 should be the same length as pLacIQ. The gels do not show this, but instead show that the sample in Lane 5 is smaller than that in Lane 6 because Lane 5 traveled further. Leland Taylor and Romina Clemente sent out an email to Pallavi Penumetcha to ask about this because she had written a paper about this.

We then unloaded the 0.8% agarose gel and then placed this in the BioRad machine under the same protocol as above. The following image is the 0.8% Agarose.

[[Image:2009-06-09_15hr_21min.jpg]]

We observed that the bands in Lane 1 (which is now at the top) are examples of high intensity bands. The plasmids contained in the original well are twice the intensity of the insert.

In Lane 5, however, we observed several different bands. This could be due to the extra DNA that was sitting on the top of the well. We decided to re-expose the gel to get better observations on Lanes 3 and 4 because the bands were not as intense. We anticipated seeing a band at about 250 bp because this was the size of the insert (plus extra bp). After re-exposing the gel, we did not find any difference and still did not see any bands in these lanes. We have decided to redo the gel on the part S03511 and so we prepared the master mix with that DNA and incubated it so that we could run the gel tomorrow.

We also cut out the "empty" plasmids from the gel so that we could use this as vectors for the coding sequence of the suppressor tRNAs.

Tomorrow we will be planning out specific tasks for people once the oligos arrive.

==Wednesday, June 10, 2009==

We began this morning by making the agarose gel with the same protocol as yesterday. This time, since we are just re-doing both S03511 inserts, we will have a 1.8% agarose gel.

'''OUTLINE: WHERE DO WE STAND?'''

Yesterday
a. Isolated empty plasmids
b. Verified most inserts
c. Waiting for:
>tRNA oligos
>Primers
a. Will amplify the reporter gene in the plasmid itself

Next?
a. When Primers Arrive
> Amplify the reporter genes with primers (PCR takes ~3 hours)
> Calculate how much reporter plasmid we will need (Start with 50x more than we actually will need)
> Cut amplified sequence (EcoR1 and BamH1/Nco1) then purify what we need
> Cut reporter plasmid (EcoR1 and BamH1/Nco1) then purify what we need
> Ligate amplified sequence and reporter plasmid
b. When tRNA oligos arrive
> Calculate how much of each oligo we will need (takes a while...)
> "Boil and cool" tRNA oligos together
> Ligate fragments (dsDNA) into plasmids we isolated yesterday (~5 minutes)
> Transform the plasmids (~20 minutes)

* LOOK THROUGH LIGATION PROTOCOL (How to assemble genes)
* LOOK THROUGH TRANSFORMATION PROTOCOL

''What we need to get done today'':
1. Prepare the control for the Tet Resistance by BioBricking the part pLac+RBS (S03511) with the TetA (J31007).
2. Check the part number for RFP and also for pLac+RBS+RFP
3. Read through ligation protocol
4. See how many nanograms of plasmid we will need for the tRNAs and for the inserts and multiply this number by 50.
> Check if we have enough plasmids from our mini-prep
5. Purify the plasmids from the gel

After we made this outline, Leland, Olivia and I analyzed the gel we ran this morning and did not see a more than one band in the 2 lanes that contained DNA from S03511 (image below). Since we did not see multiple bands in yesterday's gel either, we conclude that there is something wrong with the DNA rather than with the restriction enzymes that we used to cut it. We concluded that there is probably not an insert in this plasmid both the part and the plasmid are useless.

[[Image: 2009-06-10 10hr 30min.jpg]]

Now, we do not have an immediately accessible promoter. We have decided to order the pBAD promoter from Missouri Western and we will attach the RBS+Tet to this plasmid. Our insert for this will be the larger gene because it is easier to manipulate in the gel than the small gene.

Alyndria Thompson and Romina Clemente are figuring out the size of the plasmids including the reporter genes.

After lunch we performed several steps to isolate and purify the gel to get the empty plasmids will be the vectors for the tRNA DNA. Then, we used the NanoDrop to assess the purity of our DNA. We found that the plasmids were more pure than we had expected. The following values were obtained:

Abs 1.850
A-260 10 mm path 0.172
A-280 10 mm path 0.075
260/280 2.28
260/230 0.09

We then followed the protocol for preparing the agar plates. We put the plate with the agar solution in the Autoclave.

While the solution was in the autoclave, we discussed that we don't really need a promoter in front of RBS+Tet for the gene to be expressed because Tet expression is so strong that the gene would translate without a promoter. However, it would be a good idea to have the promoter because if it was possible to reinforce the translation of the reporter protein, we wanted to do that.

We found that there were some tubes in the lab fridge that contained the pBAD and pLac promoters with RBS. We decided to use these as the promoter and RBS for our experiments. The pLac promoter will be used for RFP and the pBad promoter used with Tet Resistance. We decided to use the pBad with Tet because it is weaker in comparison to pLac. As expression of the Tet gene can be toxic to a cell, we do not want to overexpress the gene with a stronger promoter.

Once the agar solution was sterilized in the Autoclave, we removed it, cooled it, and pipetted the antibiotic resistance into it. We then poured the solution into the plates, removed any bubbles, and let the gel form.

Alyndria Thompson and Romina Clemente concluded that we had more than enough recieving vectors for both the RFP and Tetracycline genes. Since we need 50 nanograms, it is wise to have at least 50 times that much that we can work with, so we need at least 2,500 ng.

RFP 1: 77.6 x 4 = 310.4 ng/uL
x 20 uL = 3,272 ng
RFP 2: 59.5 x 4 = 238 ng/uL
x 20 uL = 4,760 ng
Tet: 40.9 x 4 = 163.6 ng/uL
x 20 uL = 3,272 ng

According to these calculations we have more than 50 times what we need for both instances of RFP and Tet.

Tomorrow, we can prepare the Master Mix for the digestions of both reporter plamids and then add the restriction enzymes once they arrive. Then, we can follow through with the rest of the plans we had made above in the section titles "Next?".

==Thursday, June 11, 2009==

We began this morning by calculating how many uL we would need for each of the mixes for digesting our reporter plasmids. We decided to double the final volume of each mix to 40uL instead of 20uL. Since we knew the concentration of each plasmid and also knew we needed 2500 ng, we cross multiplied to find the uL of DNA we would need for each.

Concentration Calculations:
310.4 ng/1 uL = 2500ng/x uL
x = 8.05 uL

Here is a chart indicating how much of each we used for the 2 mixes.

[[Image: DoubledigestRFPTetR.png]]

On the Promega website, we found a list of compatible buffers for double digestions. Buffers E and H are compatible for the Tet plasmid and RFP plasmid digestions respectively. We fortunately have both buffers in stock.

[[Image:BamEcoR1.png]] [[Image:NcoEcoR1.png]]

We waited for our waited for the restriction enzymes to arrive and in the meantime, we transformed the plasmids with pLac+RBS, pBad, and pBad+RBS into ''E.coli'' cells. We want the ''E.coli'' cells to replicate the plasmids with these genes because we have a limited amount of genes. [http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_Transformation.html Zippy Plasmid Transformation]

Once our oligos and restriction enzymes arrived, we got to work right away!

'''PCR (Polymerase Chain Reaction)'''

We calculated how much plasmid we will need for the PCR. We decided to dilute the concentration of our plasmids a hundred fold so that we could get pipetable amounts.

''Change in Concentrations''
310.4 ng/uL x 1/100 (dilution factor) = 3.104 ng/uL (RFP)
163.6 ng/uL x 1/100 (dilution factor) = 0.164 ng/uL (Tet)

''Example shown with RFP''
3.104 ng/1 uL = 1ng/ x uL, x = .32
For Tet, x = .61

We also calculated how much of each primer we would need with the following equation:

C1V1 = C2V2
(100 uM) (x) = (1 uM)( 100 uL)
x = 1 uL

[[Image: screen-capture-8.png]]

Each of the 5 of us made had 2 tubes (one for each reporter protein) because there were 5 different forward primers.To each tube, we added the template DNA (either RFP or Tet), water, the respective reverse primer (either RFP or Tet), and the forward primer depending on which 5mer we had chosen. My 5mer was CCAUC. Finally, we added the Master Mix last because this contained the enzyme (DNA polymerase) that would amplify our desired sequence.

[[Image: Pcrtubes.png]]

We followed the temperature cycles so that PCR could occur. The annealing temperature needed to be 5C less than the smallest melting temperature of the primers. Since the reverse primer of RFP had a melting temp of 66C, we set the temperature of the 3rd cycle on 61C.

[[Image: Temperaturecycle.png]]

'''tRNA Oligo Assembly'''

We needed to calculate the concentration of each of the oligo before carrying out the assembly. The stock concentration of each of the oligo was 100 uM. Since we wanted to have 5uM of the oligo in the test tube, and we know that final volume of the test tube is 20 uL, we just cross multiplied to find the amount we needed to add to the tubes to get this concentration.

(100uM)x = (5uM)(20uL)
x = 1 uL

We added 1 uL of each oligo to the tubes.

We had six total tRNAs we needed to assemble so each of us took one FML (one person took two) and to each tube we added 1uL of each oligo for a total of 7uL, 2uL of buffer, and 11uL of water for a total volume of 20 uL. Five of the oligos were constant and 2 varied for each of the different tRNAs. So everyone pipetted the same 5 oligos and everyone also had 2 unique oligos to add. When all of the oligos were added and the tubes ready, we boiled the tubes at 50C for 5 minutes. We then allowed the tubes to remain in the water to cool very slowly overnight so that the annealing process could take place.

The last thing we did at the end of the day was finish off our plasmid digestion that we had begun in the morning. We added the buffer, DNA, water, and restriction enzymes and placed this in the incubator for overnight digestion.

'''To Do''':

1. Isolate our desired dsDNA from the PCR.
2. Verify our tRNA inserts and eventually transform these into plasmids.
3. Check our agar plates to see colonies that have replicated our pLac+RBS, pBAD+RBS, and pBAD genes.

==Friday, June 12, 2009==

This morning, we began by outlining the main items on today's agenda.

1. tRNAs: Ligation, Transformation
2. Digestion reporters (run all of it and gel purify)
3. Transformed cells
4. PCR: Gel run an aliquot to verify (5uL)

We used the equation given by the Ligation protocol to calculate how many nanograms of insert we would need. Initially, we started out calculated the MW average of the 9 and 10 anticodon loops of tRNAs which were 15828.23 ug/umols and 159016.6 ug/umols respectively. We then saw, however, that these would not be helpful values in the process. Instead we started out with the equation below to find the final ng of insert we would need for the ligation.

[[Image: ligationcalculations.png]]

We calculated our initial concentration in ng/uL of insert as shown below.

[[Image: ligationequation.png]]
X nanograms of insert = [(2)(144bp)(50 ng linearalized plasmid)]/2038bp
X = 7.0657 ng of insert

We wanted to find the amount of uL of insert we would need to use. We diluted the initial concentration 50x because we wanted a pipettable amount of insert. Since we knew we wanted the final amount of nanograms to be 7.0657, we calculated as shown below:

''DILUTION'': 555.48/50 = 11.012
''CALCULATION'': 11.012 ng/ 1uL = 7.065 ng/ x uL
x = .64 uL of insert

After we had done these calculations, half of the group started to carryout the ligation of the tRNA inserts into empty plasmids. We needed 7 ligations total: 6 were the tRNAs we needed, while the last one is a negative control. These are the final amounts of what we used:

[[Image: Trnaligation.png]]

The other half of the group (which included me) prepared the agarose gels so that we could run gels for the digested reporters and the PCR inserts. We calculated that the digested reporters needed an agarose of 4% and the PCR reporters needed an agarose of 1.45%. We allowed this gel to form for about 45 minutes to an hour. We prepared both the gels and allowed them to run through lunch.
After lunch, we took pictures of our gels and noticed that the third lane in the PCR gel (third lane from the top in the bottom photo) did not develop a band. The lane was supposed to contain the CUAGU insert for Tetracycline Resistance.

[[Image: 2009-06-12 15hr 54min.jpg]] [[Image:2009-06-12_16hr_07min.jpg]]

Romina Clemente and I decided to repeat this PCR using the TetResistance and RBS+TetResistance plasmids as templates. We labeled J31007 as "2*" and S03710 as "2!". We had to recalculate how much template DNA to use. We let the PCR run overnight. We also verified that the digestion fragments (both the plasmid and the insert) on the other gel were the correct size. The plasmids, which are the brighter fragment in the gel with only 2 lanes, contain the plasmid base pairs along with the rest of our reporter genes. We isolated these plasmids from the gel today and stored them for purification on Monday.

In the meantime, other members of our lab had finished the ligation of the tRNA genes to the empty plasmids and were now transforming these into competent cells. They placed the agar plate with these cells in the fridge overnight.

We also observed the transformations that we had performed yesterday and noticed that I13453 did not form any colonies. J04500 and J3102 did form colonies.

To Do:
1. PCR: Dig, Ligate, Transform
Clean and concentration column, digest, clean and concentration column, ligate.
2. Gel purification of our digestion plasmids
3. Check if colonies have grown for our tRNAs

==Saturday, June 13, 2009==

Alyndria Thompson came in on Saturday morning to remove the tRNA plates and put the PCR reactions in the fridge.

'''tRNA Colonies'''

She counted the colonies of tRNA on each plate.

[[Image:TRNA_colonies_6_13_09.png]]

She placed the plates in the large fridge on the top shelf at 9:23 am. She talked to Dr. Campbell who said we will have to try to "work them up".

'''PCR'''

Products were placed in orange rack of smaller fridge. [2*, 2!]

==Sunday, June 14, 2009==

'''Second Ligation and Transformation of tRNA inserts'''

We decided to re-ligate and re-transform all of the tRNA inserts because we did not get very good yields of colonies from the transformations.
We are using the same measurements for this ligation as we had used on Friday.

[[Image: Trnaligation.png]]

After ligation, we transformed the cells. Using the following measurements, we transferred the competent cells into new tubes.

40uL of competent cells
10uL of ligation mixture
60uL of LB media

After spreading the mixture onto the agar plates, we incubated the plates at 10:56 pm.

We also stored liquid cultures of the colonies that greater than a 2:1 variable:negative control ratio just in case we wanted to use them for mini preps in the future. These included #1 (3 samples), #4 (2 samples), #5 (1 sample), and #6 (2 samples).

'''To Do''':
1. Count colonies obtained from second transformation
2. Clean PCRs: Dig, ligate, transform
3. Run gel for 2 Tet PCRs, clean
4. Gel purify receiving vector

IGEM Notebook

2009-06-15T04:00:28Z

ShPunjabi: /* Sunday, June 14, 2009 */

==Wednesday, June 3, 2009==

Romina Clemente and I are trying to find suitable reporter proteins to use. Yesterday, Leland Taylor and Alyndria Thompson were working on ways to insert the gene sequences into the plasmid. Upon seeing how they wanted to manipulated the reporter gene to include the logical clauses, we came up with a few criteria for the reporter genes we would use. The following criteria for genes are listed in order of the broadest aspect to look at to the narrowest aspect:
a) Doesn't contain restriction sites for the 4 restriction enzymes (EcoR1, Xbal, Spel, Pst1) used to cleave the Biobrick part out of the plasmid.
b) Contains 6 cutter restriction sites.
c) These restriction enzymes aren't blunt (cleave straight down at one spot).
d) These restriction sites are close to thge 5' (beginning) end of the sequence.
e) These enzymes are easiest to work with and cheapest.
We are finding the part numbers of the reporter genes we want to use (antibiotic resistance, fluorescence, LacZ) through our own GCAT because we know these ones work. We are then locating these parts on the parts registry [1] website. We copied and pasted the gene sequences we obtained from the registry onto the ApE software [2]. From here, we were able to generate a genetic map of each gene that outlined each restriction site that fit our criteria. We put each genetic map, alongside the part number used, into a Word document.

http://gcat.davidson.edu/GcatWiki/images/0/0e/Restriction_Site_Mapping_on_Reporter_Genes.doc

Later on in the day, we decided that the best way to test for suppression would involve placing the 5mers at the beginning of the reporter gene instead of inserting them into the reporter gene. To do this, we decided that the gene along with its start codon need to be expressed after the 5mer. We will insert a BioBrick plasmid with a 5mer and start codon that will be incorporated before the gene:
RBS-6-8nt-ATG-5mer--ATG-gene

Leland Taylor assembled the oligos that we will need for the BioBrick pieces.

In order to test whether the reporter protein would be expressed regardless of the 5mer being suppressed or not, we would need to remove the suppressor tRNAs from the cell.

Meanwhile, I helped figure out the coding sequence that we will need for the suppressor tRNAs that will bind to the 5mers. We decided that we would be using the following suppressor tRNAs on the Davidson side: CUAGU, CCCUC, CGGUC, CCAUC, and CCACU. We searched through several papers, with little result and finally emailed Dr. Christopher Anderson of UC Berkeley to request the DNA sequence. He emailed us back with a generic DNA code that we could use to create the tRNA; however, for each suppressor tRNA we would use, we would need to change base pairs in the anticodon loop.

The length of the tRNA is 92 nt. The anticodon loop is comprised of 9 nt. Once we have BioBricked the DNA sequence with 4 restriction sites and supplemental nucleotides, the length of the entire gene is 144 nucleotides. This is made up of the 92 nt +22 nt on each side of the sequence (44) + 4 nt on each side of the restriction ends (8).

At the end of the day, we decided to use tetracycline resistance and RFP as our reporter proteins for this tester experiment.

==Thursday, June 4, 2009==

This morning, I began the day by learning how to use the PEARL prgram. Olivia Ho-Shing sat with me and walked me through the program script. And then, Leland Taylor and Shashank Suresh had a problem they wanted us to solve. Leland explained that we need to make sure there is a stop codon in the gene if we fail to suppress the 5mer. The stop codon needs to be before we find another 5mer. They also wanted us to run through both the RFP and Tet Resistance genes to make sure that they did not contain any of the anticodon sequences from our 5 frameshift suppressor tRNAs so as to throw the translation out of frame. They further wanted us to confirm that if we do have successful suppression, there are no stop codons in the middle of the RFP and Tet Resistance genes. PEARL did not find any matches for the suppressor tRNAs within genes. We found 3 matches for stop codons UAA. Two of these occured at the end of the Tet and one at the end of RFP, where they should normally be to end translation into a protein.

Following this, Olivia Ho-Shing and I tried to determine how we can add BioBrick ends to our single stranded DNA sequences that code for the suppressor tRNAs. Our ultimate goal is to find complements for these single strands so they can be put into plasmids. We found the standard prefixes and suffixes for sequences that do not contain ATG (becuase this sequence is a functional RNA and will not be translated) and we placed these before and after the altered 92bp sequence that Dr. Anderson provided us with. The prefixes and suffixes are necessary because they are the extra nucleotides that will be mimicking a "restriction enzyme cutting site". The single strand appeared as below after the Bio Brick prefixes and suffixes were added:

22ntPREFIX--32nt--VARIABLE ANTICODON LOOP--51nt--21ntSUFFIX

We used the suppressor codon CUAGU as our example and placed its appropriate anticodon loop in the sequence. We put this into the lancelator that would give us the other strand as well as the oligos we would need for ideal construction of this double stranded DNA fragment to put into the plasmid. We recieved a total of 4 oligos, 2 of which were variable and 2 of which were constant for all 6 different suppressor tRNAs. Using this double strand, we simulated a restriction enzyme digestion by EcoRI and PstI. We took off the G at both ends of the top strand and then removed the first five nucleotides on each end of the bottom strand. This made the top strand longer than the bottom one. We then proceeded to take the two variable strands and changed the anticodon loop nt for each suppressor tRNA we used.

Around lunchtime, we encountered a problem with the BioBrick scar in the "ATG-5mer subpiece" of the test project. It turns out that after annealing the sticky ends of the Xba1 and Spe1 sites, the scar created had a stop codon TAG (UAG) that was in frame with the suppressed codon. When the Xba1 site on insert anneals to the Spe1 site on the plasmid (the green portions), we get a scar that reads TACTAG.
[[Image:screen-capture.png]]

For the afternoon, Olivia Ho-Shing and I finished assembly of the oligos of the suppressor tRNA codes. We assembled the document below:

http://gcat.davidson.edu/GcatWiki/images/5/5e/TRNAoligostoOrder.doc

We were assigned tasks of creating "cartoons" to illustrate the phenomenon that had occurred. No one could successfully do this however, because we could not understand how the piece had formed. The parts registry website provided wrong information on the restriction site of Spe1 which slowed our efficiency. This project turned into an effort to try to find two other different restriction enzymes that have complementary sticky ends other than Xba1 and Spe1. We want to get rid oc the scar that contains TACTAG.

We ended up deciding that a hybrid of two ideas was needed: restriction enzymes would need to be used to make several reporter gene sequences with different beginnings dependending on the suppressor tRNA sequence needed.

==Friday, June 5, 2009==

Our goal for this morning was to discuss the hybrid PCR approach to assemble the part that comes before the gene we wanted to express. We decided that we would have a BioBrick prefix and a 5mer followed by a sequence of nucleotides whose identity was up to us. Using PCR we would get the second double strand if we have the following:

BioBrick Prefix--ATG--5mer--gene until we get to restriction site

The PCR primer would attach to the ATG and the 5mer and would replicate the complementary base pairs to make a double stranded DNA sequence. As several of us were confused about PCR methods, we looked at the online link provided by Alyndria Thompson's link to the PCR interactive website which was very helpful. We also did some research to discover that the restriction enzyme BseRI (which is contained in the YFP that we wanted to alter) is not compatible with any other restriction enzyme. This is good because we did not want to plasmid containing this gene to close up with Xba1 once we digested it.

The team experienced some confusion with the hybrid idea and spoke to Clif Davis of Missouri Western University on the phone. He told us that there will be one part that is a fragment of the beginning of some nucleotides as a restriction cleaving site, ATG, the logical clause, and the start of the YFP gene that will be placed into a plasmid containing a space. To the left of the space will be the BioBrick Prefix and the right of the space will contain the rest of the genes in the YFP. Therefore, we decided not to alter any BioBrick ends and just look at restriction sites at the beginning of reporter proteins. The small fragment that will be placed into the plasmid will be made by PCR. The PCR will attach a primer onto the ATG and 5mer and replicate.

While we were discussing this, we expanded upon the idea that we can do whatever we want with the "suffix" portion between the 5mer and the beginning of YFP. We decided it would be best to remove ATG and add the first 5 amino acids and the restriction enzyme site. Along with this, if we have the 5mer coding for a certain amino acid (ie methionine) we could replace another codon that codes for methionine WITH that 5mer so long as this codon is contained within the first 15 nt. This way, we would not be altering the protein.

Olivia Ho-Shing came up with an idea of cascading PCR effect for longer logical clauses. Post translational modification of the protein was another one of her ideas. This applies because although we may code for the protein in a given way, the protein may modify or fix itself up to perform a slightly different function in its tertiary form.

Dr. Campbell suggested that we can look up restriction enzymes 400-500 base pairs into the reporter protein we would like to use; however, we have more flexibility in our procedure if we choose more "hardy" cutters. So instead, we could cut with EcoRI instead of with Xba1.

During the afternoon, we set about three tasks:
1) Which primers to use
2) Which promoters to use
3) Which restriction enzymes and how they will determine use of reporter proteins

Olivia Ho-Shing and I began looking up which restriction enzymes were strong so we could base our reporters on that. We began with Tet and RFP since those were the two reporters we had started out with choosing. I found a website that provided enzyme cleavage efficiency based upon the proximity of the site to the end of the PCR fragment that contains it. We discovered the following based upon the attached documents:

http://gcat.davidson.edu/GcatWiki/images/8/8f/Restriction_Enzyme_Cleavage_Efficiency.doc
http://gcat.davidson.edu/GcatWiki/images/9/9a/07Jun09_RE_Cutting_Efficiency_oh.doc

We decided that these two reporters contained efficient enzymes in a good quantity to choose from. On Monday, we will look through these enzymes once more and finalize which enzymes we would like to use with RFP and Tet.

==Sunday, June 7, 2009==

We did preparations for the mini prep this evening. The following notes are courtesy of Olivia Ho-Shing.

1. Sterically transfer ~2mL LB+AMP (AMP are ampicillin resistant plasmids) *Never put fluid back into bottle once it has entered pipette*
2. Use forceps to grab toothpick, scrap some frozen cells, rub in tube with top

'''Tubes'''

S03511 (2)

K091111

B0030

I715039

K091112

I715039

J31007 (2)

B0034

E1010

S03710

==Monday, June 8, 2009==

We reviewed the PCR process this morning as well as the restriction enzyme cleavage process.

Then, Leland Taylor, Olivia Ho-Shing and I all worked on converting the tRNA oligos we made to the template that Missouri Western used. The oligos we had previously made were different from the ones that Dr. Anderson had originally emailed us because further communication between Dr. Anderson and Missouri Western resulted in a better template.

We tried several times to minimize the number of oligos while keeping their individual lengths under 80bp. We managed to do this in the end by creating 7 oligos, 2 of which are variable between the suppressor tRNAs.
We reviewed the following document and finalized the oligos that we needed to order for the frameshift suppressor tRNAs. We decided to keep both the 10bp and 9bp anticodons of CCAUC.

I also looked up the amino acids that corresponded to the FS (frameshift suppressor) tRNAs for future use:

1. CUAGU = Leu
2. CCCUC = Pro
3. CGGUC = Arg
4. CCACU = Pro
5. CCAUC* = Pro
*9 and 10 bp anticodons

Also, we have decided to use NcoI as the restriction enzyme for RFP. This cleavage site occurs 419 bp into the gene. We have decided to use BamHI as the enzyme for Tetracycline Resistance and the cleavage site occurs 290bp into the gene. Both of these enzymes are very powerful and hardy.

We also looked over Missouri Western's tRNA oligos and primer oligos which we approved.
Our oligos were also approved by Missouri Western and Dr. Campbell ordered them while we supervised.

In the afternoon we did the miniprep. The mini prep directions were as appear in the link below.

http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_MiniPrep.html

We then worked with the Nano Prep to dilute our solution of DNA to an absorption of 1.5 and then we made labels for the tubes. The chart below shows relevant information we obtained from the NanoDrop for each of the reporter plasmids.

[[Image: Screen-capture-6.png]]

==Tuesday, June 9, 2009==

'''Preparations for restriction enzymes digestion of plasmids'''

Purpose: To determine the size of the plasmid inserts and ensure that plasmids are what we extracted from the cells during the mini-prep.

Things we need:
DNA ~3uL
Buffer 10x (Want to dilute to 1x) so add 2uL
Enzyme 1 ~1uL
Enzme 2 ~1uL
Water ~13uL
Final Volunter about 20 uL.

Need to choose the right buffer for the restriction enzymes. All buffers come in 10x (10 times more concentrated than what you want it to be when you use it). [http://gcat.davidson.edu/GcatWiki/index.php/Davidson_Missouri_W/Double_Digest_Guide Buffer Website]

Total volume of enzymes has to be 10% or less than final volume (most volume 2uL). Barely submerge the pipet tip into the solution when pipeting the enzymes.
Star (*) activity: Restriction enzyme stops cutting specifically and starts cutting everywhere.

DNA can't be seen by itself so we add EtBr (the bigger the insert, the more will bind) so bigger strands appear brighter. samples.

The order of entry doesn't matter, but make sure to enter the enzymes last.

Will have lots of labeled tubes and will put 3uL of appropriate plasmids into the tubes.
In the meantime, we'll make a "Master Mix" of all the other ingredients in a bigger tube. Always make enough volume for one extra tube.

Enzymes usually have an optimal temperature of 37C. We will let this reaction go for an hour and then we will run it on a gel.

'''Gel Electrophoresis Protocol'''

Website online for ideal gel concentration for resolving different sizes of molecules [http://gcat.davidson.edu/iGEM08/gelwebsite/gelwebsite.html Gel Concentration Website].
See protocol on the Davidson/Missouri Western Wookie for running molecules in the gel and how to digest with restriction enzymes.

'''What's Next?'''

1. 5' end additions
a. Using PCR
b. Cut at the restriction sites EcoR1 and either Bam H1 or Nco1
c. Cut plasmid and reporter with same restriction enzymes as above
d. Run a gel and purify "keeper" DNA (Ligate transform, screen, sequence)
e. Then, we take the "keeper" DNA and combine it with the insert from part

We can have steps d and e ready in advance.

2. tRNAs
a. Assemble the oligos into genes (Requires lots of calculations beforehand, but the actual steps )
b. Plasmids cut up with EcoR1 and Pst1 '''WE CAN USE THE "WASTE" PLASMID FROM THE RESTRICTION ENZYME DIGEST'''
c. Gel purify the plasmids (Ligate transform, screen, sequence)

The following tables include Master Mix measurements, agarose gel concentrations and buffers.
http://gcat.davidson.edu/GcatWiki/images/0/08/Shamitapic3.png
http://gcat.davidson.edu/GcatWiki/images/d/d1/Shamitapic4.png
http://gcat.davidson.edu/GcatWiki/images/6/6f/Shamitapic5.png

We made the Master Mix with our DNA and put it in the incubator at 10:40 AM.

'''Preparing the Gels'''

Now, we will be making 2 gels, 1 at 0.8% and 2.5% (weight to volume percent). The gel volume is 60mL.

'''Definition of 1% solution:
1 gram of agarose/ 100mL buffer'''

So for a 0.8% solution: 0.8 grams agarose/100mL buffer AND we need 60mL (fixed volume for casts run) so multiply that by 0.6.
Final concentration:

.48 grams agarose/60mL buffer

1 uL of EtBr stock for 60mL of gel.
EtBr is in the buffer so it is constantly replenishing itself even though it is moving toward one end in order to make bands on the other end visible.

After Dr. Campbell's demonstration, we each helped prepare a 0.8% buffer solution using the agarose powder, buffer and EtBr. We mixed together the first two substances in a 200mL Erlenmeyer flask and heated it for 120 seconds in the microwave. We then added EtBr and poured the gel solution into the gel cast.

'''Agar Plate'''

After lunch, we grew some more cells for each of the 11 parts in an agar plate labeled "LBAMP-AMC-9 June" so that so would have extra cells that could be stored for up to 2 weeks.
We made small circles on the agar plate and numbered them 1-11. The numbers corresponded to the following parts. We put the plate in the incubator overnight to let the cells grow.

1. B0030 AMC
2. K091111
3. S03710
4. I715039 OEH
5. S03511 ORH
6. I715039 AST
7. B0030 SDP
8. K091112
9. J31007 LJH
10. J31007 REC
11. S03511

'''Unloading the Gels'''

The following shows which parts we entered in which gels:

[[Image: screen-capture-2.png]]

We unloaded the 2.5% agarose first and after observing the bands under a UV light, put the gel into the BioRad machine to observe the bands. The image below is of a Molecular Weight Marker that shows the number of bp in control fragments of DNA according to the distance they travel down the gel.

[[Image: DNA_molecular_weight_marker_gel.png]]

We obtained the following printout for the first gel that was 2.5% Agarose.
[[Image: 2009-06-09_14hr_44min.jpg]]

The bands in the first well at the bottom are those of the molecular weight marker. We skipped the lane above (to the right) of the MWM and entered the rest of the inserts as described in the above table. A light band is visible for each of the 4 other wells. In Lanes 3 and 4 we concluded we are supposed to have approximately 45 bp because the insert is 15bp + 30 bp on either side of the insert (Xba1 and Spe1 restrictions sites + sticky ends + extra bases). We used the same logic for Lanes 5 and 6 which are supposed to contain approximately 86 base pairs.

One discrepancy in this gel, however, is that Lanes 5 and 6, which according to their labels respectively contain the LacIQ promoter and the Lac IQ1 promoter. According to the information online at the parts registry website, pLacIQ1 should be the same length as pLacIQ. The gels do not show this, but instead show that the sample in Lane 5 is smaller than that in Lane 6 because Lane 5 traveled further. Leland Taylor and Romina Clemente sent out an email to Pallavi Penumetcha to ask about this because she had written a paper about this.

We then unloaded the 0.8% agarose gel and then placed this in the BioRad machine under the same protocol as above. The following image is the 0.8% Agarose.

[[Image:2009-06-09_15hr_21min.jpg]]

We observed that the bands in Lane 1 (which is now at the top) are examples of high intensity bands. The plasmids contained in the original well are twice the intensity of the insert.

In Lane 5, however, we observed several different bands. This could be due to the extra DNA that was sitting on the top of the well. We decided to re-expose the gel to get better observations on Lanes 3 and 4 because the bands were not as intense. We anticipated seeing a band at about 250 bp because this was the size of the insert (plus extra bp). After re-exposing the gel, we did not find any difference and still did not see any bands in these lanes. We have decided to redo the gel on the part S03511 and so we prepared the master mix with that DNA and incubated it so that we could run the gel tomorrow.

We also cut out the "empty" plasmids from the gel so that we could use this as vectors for the coding sequence of the suppressor tRNAs.

Tomorrow we will be planning out specific tasks for people once the oligos arrive.

==Wednesday, June 10, 2009==

We began this morning by making the agarose gel with the same protocol as yesterday. This time, since we are just re-doing both S03511 inserts, we will have a 1.8% agarose gel.

'''OUTLINE: WHERE DO WE STAND?'''

Yesterday
a. Isolated empty plasmids
b. Verified most inserts
c. Waiting for:
>tRNA oligos
>Primers
a. Will amplify the reporter gene in the plasmid itself

Next?
a. When Primers Arrive
> Amplify the reporter genes with primers (PCR takes ~3 hours)
> Calculate how much reporter plasmid we will need (Start with 50x more than we actually will need)
> Cut amplified sequence (EcoR1 and BamH1/Nco1) then purify what we need
> Cut reporter plasmid (EcoR1 and BamH1/Nco1) then purify what we need
> Ligate amplified sequence and reporter plasmid
b. When tRNA oligos arrive
> Calculate how much of each oligo we will need (takes a while...)
> "Boil and cool" tRNA oligos together
> Ligate fragments (dsDNA) into plasmids we isolated yesterday (~5 minutes)
> Transform the plasmids (~20 minutes)

* LOOK THROUGH LIGATION PROTOCOL (How to assemble genes)
* LOOK THROUGH TRANSFORMATION PROTOCOL

''What we need to get done today'':
1. Prepare the control for the Tet Resistance by BioBricking the part pLac+RBS (S03511) with the TetA (J31007).
2. Check the part number for RFP and also for pLac+RBS+RFP
3. Read through ligation protocol
4. See how many nanograms of plasmid we will need for the tRNAs and for the inserts and multiply this number by 50.
> Check if we have enough plasmids from our mini-prep
5. Purify the plasmids from the gel

After we made this outline, Leland, Olivia and I analyzed the gel we ran this morning and did not see a more than one band in the 2 lanes that contained DNA from S03511 (image below). Since we did not see multiple bands in yesterday's gel either, we conclude that there is something wrong with the DNA rather than with the restriction enzymes that we used to cut it. We concluded that there is probably not an insert in this plasmid both the part and the plasmid are useless.

[[Image: 2009-06-10 10hr 30min.jpg]]

Now, we do not have an immediately accessible promoter. We have decided to order the pBAD promoter from Missouri Western and we will attach the RBS+Tet to this plasmid. Our insert for this will be the larger gene because it is easier to manipulate in the gel than the small gene.

Alyndria Thompson and Romina Clemente are figuring out the size of the plasmids including the reporter genes.

After lunch we performed several steps to isolate and purify the gel to get the empty plasmids will be the vectors for the tRNA DNA. Then, we used the NanoDrop to assess the purity of our DNA. We found that the plasmids were more pure than we had expected. The following values were obtained:

Abs 1.850
A-260 10 mm path 0.172
A-280 10 mm path 0.075
260/280 2.28
260/230 0.09

We then followed the protocol for preparing the agar plates. We put the plate with the agar solution in the Autoclave.

While the solution was in the autoclave, we discussed that we don't really need a promoter in front of RBS+Tet for the gene to be expressed because Tet expression is so strong that the gene would translate without a promoter. However, it would be a good idea to have the promoter because if it was possible to reinforce the translation of the reporter protein, we wanted to do that.

We found that there were some tubes in the lab fridge that contained the pBAD and pLac promoters with RBS. We decided to use these as the promoter and RBS for our experiments. The pLac promoter will be used for RFP and the pBad promoter used with Tet Resistance. We decided to use the pBad with Tet because it is weaker in comparison to pLac. As expression of the Tet gene can be toxic to a cell, we do not want to overexpress the gene with a stronger promoter.

Once the agar solution was sterilized in the Autoclave, we removed it, cooled it, and pipetted the antibiotic resistance into it. We then poured the solution into the plates, removed any bubbles, and let the gel form.

Alyndria Thompson and Romina Clemente concluded that we had more than enough recieving vectors for both the RFP and Tetracycline genes. Since we need 50 nanograms, it is wise to have at least 50 times that much that we can work with, so we need at least 2,500 ng.

RFP 1: 77.6 x 4 = 310.4 ng/uL
x 20 uL = 3,272 ng
RFP 2: 59.5 x 4 = 238 ng/uL
x 20 uL = 4,760 ng
Tet: 40.9 x 4 = 163.6 ng/uL
x 20 uL = 3,272 ng

According to these calculations we have more than 50 times what we need for both instances of RFP and Tet.

Tomorrow, we can prepare the Master Mix for the digestions of both reporter plamids and then add the restriction enzymes once they arrive. Then, we can follow through with the rest of the plans we had made above in the section titles "Next?".

==Thursday, June 11, 2009==

We began this morning by calculating how many uL we would need for each of the mixes for digesting our reporter plasmids. We decided to double the final volume of each mix to 40uL instead of 20uL. Since we knew the concentration of each plasmid and also knew we needed 2500 ng, we cross multiplied to find the uL of DNA we would need for each.

Concentration Calculations:
310.4 ng/1 uL = 2500ng/x uL
x = 8.05 uL

Here is a chart indicating how much of each we used for the 2 mixes.

[[Image: DoubledigestRFPTetR.png]]

On the Promega website, we found a list of compatible buffers for double digestions. Buffers E and H are compatible for the Tet plasmid and RFP plasmid digestions respectively. We fortunately have both buffers in stock.

[[Image:BamEcoR1.png]] [[Image:NcoEcoR1.png]]

We waited for our waited for the restriction enzymes to arrive and in the meantime, we transformed the plasmids with pLac+RBS, pBad, and pBad+RBS into ''E.coli'' cells. We want the ''E.coli'' cells to replicate the plasmids with these genes because we have a limited amount of genes. [http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_Transformation.html Zippy Plasmid Transformation]

Once our oligos and restriction enzymes arrived, we got to work right away!

'''PCR (Polymerase Chain Reaction)'''

We calculated how much plasmid we will need for the PCR. We decided to dilute the concentration of our plasmids a hundred fold so that we could get pipetable amounts.

''Change in Concentrations''
310.4 ng/uL x 1/100 (dilution factor) = 3.104 ng/uL (RFP)
163.6 ng/uL x 1/100 (dilution factor) = 0.164 ng/uL (Tet)

''Example shown with RFP''
3.104 ng/1 uL = 1ng/ x uL, x = .32
For Tet, x = .61

We also calculated how much of each primer we would need with the following equation:

C1V1 = C2V2
(100 uM) (x) = (1 uM)( 100 uL)
x = 1 uL

[[Image: screen-capture-8.png]]

Each of the 5 of us made had 2 tubes (one for each reporter protein) because there were 5 different forward primers.To each tube, we added the template DNA (either RFP or Tet), water, the respective reverse primer (either RFP or Tet), and the forward primer depending on which 5mer we had chosen. My 5mer was CCAUC. Finally, we added the Master Mix last because this contained the enzyme (DNA polymerase) that would amplify our desired sequence.

[[Image: Pcrtubes.png]]

We followed the temperature cycles so that PCR could occur. The annealing temperature needed to be 5C less than the smallest melting temperature of the primers. Since the reverse primer of RFP had a melting temp of 66C, we set the temperature of the 3rd cycle on 61C.

[[Image: Temperaturecycle.png]]

'''tRNA Oligo Assembly'''

We needed to calculate the concentration of each of the oligo before carrying out the assembly. The stock concentration of each of the oligo was 100 uM. Since we wanted to have 5uM of the oligo in the test tube, and we know that final volume of the test tube is 20 uL, we just cross multiplied to find the amount we needed to add to the tubes to get this concentration.

(100uM)x = (5uM)(20uL)
x = 1 uL

We added 1 uL of each oligo to the tubes.

We had six total tRNAs we needed to assemble so each of us took one FML (one person took two) and to each tube we added 1uL of each oligo for a total of 7uL, 2uL of buffer, and 11uL of water for a total volume of 20 uL. Five of the oligos were constant and 2 varied for each of the different tRNAs. So everyone pipetted the same 5 oligos and everyone also had 2 unique oligos to add. When all of the oligos were added and the tubes ready, we boiled the tubes at 50C for 5 minutes. We then allowed the tubes to remain in the water to cool very slowly overnight so that the annealing process could take place.

The last thing we did at the end of the day was finish off our plasmid digestion that we had begun in the morning. We added the buffer, DNA, water, and restriction enzymes and placed this in the incubator for overnight digestion.

'''To Do''':

1. Isolate our desired dsDNA from the PCR.
2. Verify our tRNA inserts and eventually transform these into plasmids.
3. Check our agar plates to see colonies that have replicated our pLac+RBS, pBAD+RBS, and pBAD genes.

==Friday, June 12, 2009==

This morning, we began by outlining the main items on today's agenda.

1. tRNAs: Ligation, Transformation
2. Digestion reporters (run all of it and gel purify)
3. Transformed cells
4. PCR: Gel run an aliquot to verify (5uL)

We used the equation given by the Ligation protocol to calculate how many nanograms of insert we would need. Initially, we started out calculated the MW average of the 9 and 10 anticodon loops of tRNAs which were 15828.23 ug/umols and 159016.6 ug/umols respectively. We then saw, however, that these would not be helpful values in the process. Instead we started out with the equation below to find the final ng of insert we would need for the ligation.

[[Image: ligationcalculations.png]]

We calculated our initial concentration in ng/uL of insert as shown below.

[[Image: ligationequation.png]]
X nanograms of insert = [(2)(144bp)(50 ng linearalized plasmid)]/2038bp
X = 7.0657 ng of insert

We wanted to find the amount of uL of insert we would need to use. We diluted the initial concentration 50x because we wanted a pipettable amount of insert. Since we knew we wanted the final amount of nanograms to be 7.0657, we calculated as shown below:

''DILUTION'': 555.48/50 = 11.012
''CALCULATION'': 11.012 ng/ 1uL = 7.065 ng/ x uL
x = .64 uL of insert

After we had done these calculations, half of the group started to carryout the ligation of the tRNA inserts into empty plasmids. We needed 7 ligations total: 6 were the tRNAs we needed, while the last one is a negative control. These are the final amounts of what we used:

[[Image: Trnaligation.png]]

The other half of the group (which included me) prepared the agarose gels so that we could run gels for the digested reporters and the PCR inserts. We calculated that the digested reporters needed an agarose of 4% and the PCR reporters needed an agarose of 1.45%. We allowed this gel to form for about 45 minutes to an hour. We prepared both the gels and allowed them to run through lunch.
After lunch, we took pictures of our gels and noticed that the third lane in the PCR gel (third lane from the top in the bottom photo) did not develop a band. The lane was supposed to contain the CUAGU insert for Tetracycline Resistance.

[[Image: 2009-06-12 15hr 54min.jpg]] [[Image:2009-06-12_16hr_07min.jpg]]

Romina Clemente and I decided to repeat this PCR using the TetResistance and RBS+TetResistance plasmids as templates. We labeled J31007 as "2*" and S03710 as "2!". We had to recalculate how much template DNA to use. We let the PCR run overnight. We also verified that the digestion fragments (both the plasmid and the insert) on the other gel were the correct size. The plasmids, which are the brighter fragment in the gel with only 2 lanes, contain the plasmid base pairs along with the rest of our reporter genes. We isolated these plasmids from the gel today and stored them for purification on Monday.

In the meantime, other members of our lab had finished the ligation of the tRNA genes to the empty plasmids and were now transforming these into competent cells. They placed the agar plate with these cells in the fridge overnight.

We also observed the transformations that we had performed yesterday and noticed that I13453 did not form any colonies. J04500 and J3102 did form colonies.

To Do:
1. PCR: Dig, Ligate, Transform
Clean and concentration column, digest, clean and concentration column, ligate.
2. Gel purification of our digestion plasmids
3. Check if colonies have grown for our tRNAs

==Saturday, June 13, 2009==

Alyndria Thompson came in on Saturday morning to remove the tRNA plates and put the PCR reactions in the fridge.

'''tRNA Colonies'''

She counted the colonies of tRNA on each plate.

[[Image:TRNA_colonies_6_13_09.png]]

She placed the plates in the large fridge on the top shelf at 9:23 am. She talked to Dr. Campbell who said we will have to try to "work them up".

'''PCR'''

Products were placed in orange rack of smaller fridge. [2*, 2!]

==Sunday, June 14, 2009==

'''Second Ligation and Transformation of tRNA inserts'''

We decided to re-ligate and re-transform all of the tRNA inserts because we did not get very good yields of colonies from the transformations.
We are using the same measurements for this ligation as we had used on Friday.

[[Image: Trnaligation.png]]

After ligation, we transformed the cells. Using the following measurements, we transferred the competent cells into new tubes.

40uL of competent cells
10uL of ligation mixture
60uL of LB media

After spreading the mixture onto the agar plates, we incubated the plates at 10:56 pm.

We also stored liquid cultures of the colonies that greater than a 2:1 variable:negative control ratio just in case we wanted to use them for mini preps in the future. These included #1 (3 samples), #4 (2 samples), #5 (1 sample), and #6 (2 samples).

IGEM Notebook

2009-06-15T02:57:54Z

ShPunjabi: /* Sunday, June 14, 2009 */

==Wednesday, June 3, 2009==

Romina Clemente and I are trying to find suitable reporter proteins to use. Yesterday, Leland Taylor and Alyndria Thompson were working on ways to insert the gene sequences into the plasmid. Upon seeing how they wanted to manipulated the reporter gene to include the logical clauses, we came up with a few criteria for the reporter genes we would use. The following criteria for genes are listed in order of the broadest aspect to look at to the narrowest aspect:
a) Doesn't contain restriction sites for the 4 restriction enzymes (EcoR1, Xbal, Spel, Pst1) used to cleave the Biobrick part out of the plasmid.
b) Contains 6 cutter restriction sites.
c) These restriction enzymes aren't blunt (cleave straight down at one spot).
d) These restriction sites are close to thge 5' (beginning) end of the sequence.
e) These enzymes are easiest to work with and cheapest.
We are finding the part numbers of the reporter genes we want to use (antibiotic resistance, fluorescence, LacZ) through our own GCAT because we know these ones work. We are then locating these parts on the parts registry [1] website. We copied and pasted the gene sequences we obtained from the registry onto the ApE software [2]. From here, we were able to generate a genetic map of each gene that outlined each restriction site that fit our criteria. We put each genetic map, alongside the part number used, into a Word document.

http://gcat.davidson.edu/GcatWiki/images/0/0e/Restriction_Site_Mapping_on_Reporter_Genes.doc

Later on in the day, we decided that the best way to test for suppression would involve placing the 5mers at the beginning of the reporter gene instead of inserting them into the reporter gene. To do this, we decided that the gene along with its start codon need to be expressed after the 5mer. We will insert a BioBrick plasmid with a 5mer and start codon that will be incorporated before the gene:
RBS-6-8nt-ATG-5mer--ATG-gene

Leland Taylor assembled the oligos that we will need for the BioBrick pieces.

In order to test whether the reporter protein would be expressed regardless of the 5mer being suppressed or not, we would need to remove the suppressor tRNAs from the cell.

Meanwhile, I helped figure out the coding sequence that we will need for the suppressor tRNAs that will bind to the 5mers. We decided that we would be using the following suppressor tRNAs on the Davidson side: CUAGU, CCCUC, CGGUC, CCAUC, and CCACU. We searched through several papers, with little result and finally emailed Dr. Christopher Anderson of UC Berkeley to request the DNA sequence. He emailed us back with a generic DNA code that we could use to create the tRNA; however, for each suppressor tRNA we would use, we would need to change base pairs in the anticodon loop.

The length of the tRNA is 92 nt. The anticodon loop is comprised of 9 nt. Once we have BioBricked the DNA sequence with 4 restriction sites and supplemental nucleotides, the length of the entire gene is 144 nucleotides. This is made up of the 92 nt +22 nt on each side of the sequence (44) + 4 nt on each side of the restriction ends (8).

At the end of the day, we decided to use tetracycline resistance and RFP as our reporter proteins for this tester experiment.

==Thursday, June 4, 2009==

This morning, I began the day by learning how to use the PEARL prgram. Olivia Ho-Shing sat with me and walked me through the program script. And then, Leland Taylor and Shashank Suresh had a problem they wanted us to solve. Leland explained that we need to make sure there is a stop codon in the gene if we fail to suppress the 5mer. The stop codon needs to be before we find another 5mer. They also wanted us to run through both the RFP and Tet Resistance genes to make sure that they did not contain any of the anticodon sequences from our 5 frameshift suppressor tRNAs so as to throw the translation out of frame. They further wanted us to confirm that if we do have successful suppression, there are no stop codons in the middle of the RFP and Tet Resistance genes. PEARL did not find any matches for the suppressor tRNAs within genes. We found 3 matches for stop codons UAA. Two of these occured at the end of the Tet and one at the end of RFP, where they should normally be to end translation into a protein.

Following this, Olivia Ho-Shing and I tried to determine how we can add BioBrick ends to our single stranded DNA sequences that code for the suppressor tRNAs. Our ultimate goal is to find complements for these single strands so they can be put into plasmids. We found the standard prefixes and suffixes for sequences that do not contain ATG (becuase this sequence is a functional RNA and will not be translated) and we placed these before and after the altered 92bp sequence that Dr. Anderson provided us with. The prefixes and suffixes are necessary because they are the extra nucleotides that will be mimicking a "restriction enzyme cutting site". The single strand appeared as below after the Bio Brick prefixes and suffixes were added:

22ntPREFIX--32nt--VARIABLE ANTICODON LOOP--51nt--21ntSUFFIX

We used the suppressor codon CUAGU as our example and placed its appropriate anticodon loop in the sequence. We put this into the lancelator that would give us the other strand as well as the oligos we would need for ideal construction of this double stranded DNA fragment to put into the plasmid. We recieved a total of 4 oligos, 2 of which were variable and 2 of which were constant for all 6 different suppressor tRNAs. Using this double strand, we simulated a restriction enzyme digestion by EcoRI and PstI. We took off the G at both ends of the top strand and then removed the first five nucleotides on each end of the bottom strand. This made the top strand longer than the bottom one. We then proceeded to take the two variable strands and changed the anticodon loop nt for each suppressor tRNA we used.

Around lunchtime, we encountered a problem with the BioBrick scar in the "ATG-5mer subpiece" of the test project. It turns out that after annealing the sticky ends of the Xba1 and Spe1 sites, the scar created had a stop codon TAG (UAG) that was in frame with the suppressed codon. When the Xba1 site on insert anneals to the Spe1 site on the plasmid (the green portions), we get a scar that reads TACTAG.
[[Image:screen-capture.png]]

For the afternoon, Olivia Ho-Shing and I finished assembly of the oligos of the suppressor tRNA codes. We assembled the document below:

http://gcat.davidson.edu/GcatWiki/images/5/5e/TRNAoligostoOrder.doc

We were assigned tasks of creating "cartoons" to illustrate the phenomenon that had occurred. No one could successfully do this however, because we could not understand how the piece had formed. The parts registry website provided wrong information on the restriction site of Spe1 which slowed our efficiency. This project turned into an effort to try to find two other different restriction enzymes that have complementary sticky ends other than Xba1 and Spe1. We want to get rid oc the scar that contains TACTAG.

We ended up deciding that a hybrid of two ideas was needed: restriction enzymes would need to be used to make several reporter gene sequences with different beginnings dependending on the suppressor tRNA sequence needed.

==Friday, June 5, 2009==

Our goal for this morning was to discuss the hybrid PCR approach to assemble the part that comes before the gene we wanted to express. We decided that we would have a BioBrick prefix and a 5mer followed by a sequence of nucleotides whose identity was up to us. Using PCR we would get the second double strand if we have the following:

BioBrick Prefix--ATG--5mer--gene until we get to restriction site

The PCR primer would attach to the ATG and the 5mer and would replicate the complementary base pairs to make a double stranded DNA sequence. As several of us were confused about PCR methods, we looked at the online link provided by Alyndria Thompson's link to the PCR interactive website which was very helpful. We also did some research to discover that the restriction enzyme BseRI (which is contained in the YFP that we wanted to alter) is not compatible with any other restriction enzyme. This is good because we did not want to plasmid containing this gene to close up with Xba1 once we digested it.

The team experienced some confusion with the hybrid idea and spoke to Clif Davis of Missouri Western University on the phone. He told us that there will be one part that is a fragment of the beginning of some nucleotides as a restriction cleaving site, ATG, the logical clause, and the start of the YFP gene that will be placed into a plasmid containing a space. To the left of the space will be the BioBrick Prefix and the right of the space will contain the rest of the genes in the YFP. Therefore, we decided not to alter any BioBrick ends and just look at restriction sites at the beginning of reporter proteins. The small fragment that will be placed into the plasmid will be made by PCR. The PCR will attach a primer onto the ATG and 5mer and replicate.

While we were discussing this, we expanded upon the idea that we can do whatever we want with the "suffix" portion between the 5mer and the beginning of YFP. We decided it would be best to remove ATG and add the first 5 amino acids and the restriction enzyme site. Along with this, if we have the 5mer coding for a certain amino acid (ie methionine) we could replace another codon that codes for methionine WITH that 5mer so long as this codon is contained within the first 15 nt. This way, we would not be altering the protein.

Olivia Ho-Shing came up with an idea of cascading PCR effect for longer logical clauses. Post translational modification of the protein was another one of her ideas. This applies because although we may code for the protein in a given way, the protein may modify or fix itself up to perform a slightly different function in its tertiary form.

Dr. Campbell suggested that we can look up restriction enzymes 400-500 base pairs into the reporter protein we would like to use; however, we have more flexibility in our procedure if we choose more "hardy" cutters. So instead, we could cut with EcoRI instead of with Xba1.

During the afternoon, we set about three tasks:
1) Which primers to use
2) Which promoters to use
3) Which restriction enzymes and how they will determine use of reporter proteins

Olivia Ho-Shing and I began looking up which restriction enzymes were strong so we could base our reporters on that. We began with Tet and RFP since those were the two reporters we had started out with choosing. I found a website that provided enzyme cleavage efficiency based upon the proximity of the site to the end of the PCR fragment that contains it. We discovered the following based upon the attached documents:

http://gcat.davidson.edu/GcatWiki/images/8/8f/Restriction_Enzyme_Cleavage_Efficiency.doc
http://gcat.davidson.edu/GcatWiki/images/9/9a/07Jun09_RE_Cutting_Efficiency_oh.doc

We decided that these two reporters contained efficient enzymes in a good quantity to choose from. On Monday, we will look through these enzymes once more and finalize which enzymes we would like to use with RFP and Tet.

==Sunday, June 7, 2009==

We did preparations for the mini prep this evening. The following notes are courtesy of Olivia Ho-Shing.

1. Sterically transfer ~2mL LB+AMP (AMP are ampicillin resistant plasmids) *Never put fluid back into bottle once it has entered pipette*
2. Use forceps to grab toothpick, scrap some frozen cells, rub in tube with top

'''Tubes'''

S03511 (2)

K091111

B0030

I715039

K091112

I715039

J31007 (2)

B0034

E1010

S03710

==Monday, June 8, 2009==

We reviewed the PCR process this morning as well as the restriction enzyme cleavage process.

Then, Leland Taylor, Olivia Ho-Shing and I all worked on converting the tRNA oligos we made to the template that Missouri Western used. The oligos we had previously made were different from the ones that Dr. Anderson had originally emailed us because further communication between Dr. Anderson and Missouri Western resulted in a better template.

We tried several times to minimize the number of oligos while keeping their individual lengths under 80bp. We managed to do this in the end by creating 7 oligos, 2 of which are variable between the suppressor tRNAs.
We reviewed the following document and finalized the oligos that we needed to order for the frameshift suppressor tRNAs. We decided to keep both the 10bp and 9bp anticodons of CCAUC.

I also looked up the amino acids that corresponded to the FS (frameshift suppressor) tRNAs for future use:

1. CUAGU = Leu
2. CCCUC = Pro
3. CGGUC = Arg
4. CCACU = Pro
5. CCAUC* = Pro
*9 and 10 bp anticodons

Also, we have decided to use NcoI as the restriction enzyme for RFP. This cleavage site occurs 419 bp into the gene. We have decided to use BamHI as the enzyme for Tetracycline Resistance and the cleavage site occurs 290bp into the gene. Both of these enzymes are very powerful and hardy.

We also looked over Missouri Western's tRNA oligos and primer oligos which we approved.
Our oligos were also approved by Missouri Western and Dr. Campbell ordered them while we supervised.

In the afternoon we did the miniprep. The mini prep directions were as appear in the link below.

http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_MiniPrep.html

We then worked with the Nano Prep to dilute our solution of DNA to an absorption of 1.5 and then we made labels for the tubes. The chart below shows relevant information we obtained from the NanoDrop for each of the reporter plasmids.

[[Image: Screen-capture-6.png]]

==Tuesday, June 9, 2009==

'''Preparations for restriction enzymes digestion of plasmids'''

Purpose: To determine the size of the plasmid inserts and ensure that plasmids are what we extracted from the cells during the mini-prep.

Things we need:
DNA ~3uL
Buffer 10x (Want to dilute to 1x) so add 2uL
Enzyme 1 ~1uL
Enzme 2 ~1uL
Water ~13uL
Final Volunter about 20 uL.

Need to choose the right buffer for the restriction enzymes. All buffers come in 10x (10 times more concentrated than what you want it to be when you use it). [http://gcat.davidson.edu/GcatWiki/index.php/Davidson_Missouri_W/Double_Digest_Guide Buffer Website]

Total volume of enzymes has to be 10% or less than final volume (most volume 2uL). Barely submerge the pipet tip into the solution when pipeting the enzymes.
Star (*) activity: Restriction enzyme stops cutting specifically and starts cutting everywhere.

DNA can't be seen by itself so we add EtBr (the bigger the insert, the more will bind) so bigger strands appear brighter. samples.

The order of entry doesn't matter, but make sure to enter the enzymes last.

Will have lots of labeled tubes and will put 3uL of appropriate plasmids into the tubes.
In the meantime, we'll make a "Master Mix" of all the other ingredients in a bigger tube. Always make enough volume for one extra tube.

Enzymes usually have an optimal temperature of 37C. We will let this reaction go for an hour and then we will run it on a gel.

'''Gel Electrophoresis Protocol'''

Website online for ideal gel concentration for resolving different sizes of molecules [http://gcat.davidson.edu/iGEM08/gelwebsite/gelwebsite.html Gel Concentration Website].
See protocol on the Davidson/Missouri Western Wookie for running molecules in the gel and how to digest with restriction enzymes.

'''What's Next?'''

1. 5' end additions
a. Using PCR
b. Cut at the restriction sites EcoR1 and either Bam H1 or Nco1
c. Cut plasmid and reporter with same restriction enzymes as above
d. Run a gel and purify "keeper" DNA (Ligate transform, screen, sequence)
e. Then, we take the "keeper" DNA and combine it with the insert from part

We can have steps d and e ready in advance.

2. tRNAs
a. Assemble the oligos into genes (Requires lots of calculations beforehand, but the actual steps )
b. Plasmids cut up with EcoR1 and Pst1 '''WE CAN USE THE "WASTE" PLASMID FROM THE RESTRICTION ENZYME DIGEST'''
c. Gel purify the plasmids (Ligate transform, screen, sequence)

The following tables include Master Mix measurements, agarose gel concentrations and buffers.
http://gcat.davidson.edu/GcatWiki/images/0/08/Shamitapic3.png
http://gcat.davidson.edu/GcatWiki/images/d/d1/Shamitapic4.png
http://gcat.davidson.edu/GcatWiki/images/6/6f/Shamitapic5.png

We made the Master Mix with our DNA and put it in the incubator at 10:40 AM.

'''Preparing the Gels'''

Now, we will be making 2 gels, 1 at 0.8% and 2.5% (weight to volume percent). The gel volume is 60mL.

'''Definition of 1% solution:
1 gram of agarose/ 100mL buffer'''

So for a 0.8% solution: 0.8 grams agarose/100mL buffer AND we need 60mL (fixed volume for casts run) so multiply that by 0.6.
Final concentration:

.48 grams agarose/60mL buffer

1 uL of EtBr stock for 60mL of gel.
EtBr is in the buffer so it is constantly replenishing itself even though it is moving toward one end in order to make bands on the other end visible.

After Dr. Campbell's demonstration, we each helped prepare a 0.8% buffer solution using the agarose powder, buffer and EtBr. We mixed together the first two substances in a 200mL Erlenmeyer flask and heated it for 120 seconds in the microwave. We then added EtBr and poured the gel solution into the gel cast.

'''Agar Plate'''

After lunch, we grew some more cells for each of the 11 parts in an agar plate labeled "LBAMP-AMC-9 June" so that so would have extra cells that could be stored for up to 2 weeks.
We made small circles on the agar plate and numbered them 1-11. The numbers corresponded to the following parts. We put the plate in the incubator overnight to let the cells grow.

1. B0030 AMC
2. K091111
3. S03710
4. I715039 OEH
5. S03511 ORH
6. I715039 AST
7. B0030 SDP
8. K091112
9. J31007 LJH
10. J31007 REC
11. S03511

'''Unloading the Gels'''

The following shows which parts we entered in which gels:

[[Image: screen-capture-2.png]]

We unloaded the 2.5% agarose first and after observing the bands under a UV light, put the gel into the BioRad machine to observe the bands. The image below is of a Molecular Weight Marker that shows the number of bp in control fragments of DNA according to the distance they travel down the gel.

[[Image: DNA_molecular_weight_marker_gel.png]]

We obtained the following printout for the first gel that was 2.5% Agarose.
[[Image: 2009-06-09_14hr_44min.jpg]]

The bands in the first well at the bottom are those of the molecular weight marker. We skipped the lane above (to the right) of the MWM and entered the rest of the inserts as described in the above table. A light band is visible for each of the 4 other wells. In Lanes 3 and 4 we concluded we are supposed to have approximately 45 bp because the insert is 15bp + 30 bp on either side of the insert (Xba1 and Spe1 restrictions sites + sticky ends + extra bases). We used the same logic for Lanes 5 and 6 which are supposed to contain approximately 86 base pairs.

One discrepancy in this gel, however, is that Lanes 5 and 6, which according to their labels respectively contain the LacIQ promoter and the Lac IQ1 promoter. According to the information online at the parts registry website, pLacIQ1 should be the same length as pLacIQ. The gels do not show this, but instead show that the sample in Lane 5 is smaller than that in Lane 6 because Lane 5 traveled further. Leland Taylor and Romina Clemente sent out an email to Pallavi Penumetcha to ask about this because she had written a paper about this.

We then unloaded the 0.8% agarose gel and then placed this in the BioRad machine under the same protocol as above. The following image is the 0.8% Agarose.

[[Image:2009-06-09_15hr_21min.jpg]]

We observed that the bands in Lane 1 (which is now at the top) are examples of high intensity bands. The plasmids contained in the original well are twice the intensity of the insert.

In Lane 5, however, we observed several different bands. This could be due to the extra DNA that was sitting on the top of the well. We decided to re-expose the gel to get better observations on Lanes 3 and 4 because the bands were not as intense. We anticipated seeing a band at about 250 bp because this was the size of the insert (plus extra bp). After re-exposing the gel, we did not find any difference and still did not see any bands in these lanes. We have decided to redo the gel on the part S03511 and so we prepared the master mix with that DNA and incubated it so that we could run the gel tomorrow.

We also cut out the "empty" plasmids from the gel so that we could use this as vectors for the coding sequence of the suppressor tRNAs.

Tomorrow we will be planning out specific tasks for people once the oligos arrive.

==Wednesday, June 10, 2009==

We began this morning by making the agarose gel with the same protocol as yesterday. This time, since we are just re-doing both S03511 inserts, we will have a 1.8% agarose gel.

'''OUTLINE: WHERE DO WE STAND?'''

Yesterday
a. Isolated empty plasmids
b. Verified most inserts
c. Waiting for:
>tRNA oligos
>Primers
a. Will amplify the reporter gene in the plasmid itself

Next?
a. When Primers Arrive
> Amplify the reporter genes with primers (PCR takes ~3 hours)
> Calculate how much reporter plasmid we will need (Start with 50x more than we actually will need)
> Cut amplified sequence (EcoR1 and BamH1/Nco1) then purify what we need
> Cut reporter plasmid (EcoR1 and BamH1/Nco1) then purify what we need
> Ligate amplified sequence and reporter plasmid
b. When tRNA oligos arrive
> Calculate how much of each oligo we will need (takes a while...)
> "Boil and cool" tRNA oligos together
> Ligate fragments (dsDNA) into plasmids we isolated yesterday (~5 minutes)
> Transform the plasmids (~20 minutes)

* LOOK THROUGH LIGATION PROTOCOL (How to assemble genes)
* LOOK THROUGH TRANSFORMATION PROTOCOL

''What we need to get done today'':
1. Prepare the control for the Tet Resistance by BioBricking the part pLac+RBS (S03511) with the TetA (J31007).
2. Check the part number for RFP and also for pLac+RBS+RFP
3. Read through ligation protocol
4. See how many nanograms of plasmid we will need for the tRNAs and for the inserts and multiply this number by 50.
> Check if we have enough plasmids from our mini-prep
5. Purify the plasmids from the gel

After we made this outline, Leland, Olivia and I analyzed the gel we ran this morning and did not see a more than one band in the 2 lanes that contained DNA from S03511 (image below). Since we did not see multiple bands in yesterday's gel either, we conclude that there is something wrong with the DNA rather than with the restriction enzymes that we used to cut it. We concluded that there is probably not an insert in this plasmid both the part and the plasmid are useless.

[[Image: 2009-06-10 10hr 30min.jpg]]

Now, we do not have an immediately accessible promoter. We have decided to order the pBAD promoter from Missouri Western and we will attach the RBS+Tet to this plasmid. Our insert for this will be the larger gene because it is easier to manipulate in the gel than the small gene.

Alyndria Thompson and Romina Clemente are figuring out the size of the plasmids including the reporter genes.

After lunch we performed several steps to isolate and purify the gel to get the empty plasmids will be the vectors for the tRNA DNA. Then, we used the NanoDrop to assess the purity of our DNA. We found that the plasmids were more pure than we had expected. The following values were obtained:

Abs 1.850
A-260 10 mm path 0.172
A-280 10 mm path 0.075
260/280 2.28
260/230 0.09

We then followed the protocol for preparing the agar plates. We put the plate with the agar solution in the Autoclave.

While the solution was in the autoclave, we discussed that we don't really need a promoter in front of RBS+Tet for the gene to be expressed because Tet expression is so strong that the gene would translate without a promoter. However, it would be a good idea to have the promoter because if it was possible to reinforce the translation of the reporter protein, we wanted to do that.

We found that there were some tubes in the lab fridge that contained the pBAD and pLac promoters with RBS. We decided to use these as the promoter and RBS for our experiments. The pLac promoter will be used for RFP and the pBad promoter used with Tet Resistance. We decided to use the pBad with Tet because it is weaker in comparison to pLac. As expression of the Tet gene can be toxic to a cell, we do not want to overexpress the gene with a stronger promoter.

Once the agar solution was sterilized in the Autoclave, we removed it, cooled it, and pipetted the antibiotic resistance into it. We then poured the solution into the plates, removed any bubbles, and let the gel form.

Alyndria Thompson and Romina Clemente concluded that we had more than enough recieving vectors for both the RFP and Tetracycline genes. Since we need 50 nanograms, it is wise to have at least 50 times that much that we can work with, so we need at least 2,500 ng.

RFP 1: 77.6 x 4 = 310.4 ng/uL
x 20 uL = 3,272 ng
RFP 2: 59.5 x 4 = 238 ng/uL
x 20 uL = 4,760 ng
Tet: 40.9 x 4 = 163.6 ng/uL
x 20 uL = 3,272 ng

According to these calculations we have more than 50 times what we need for both instances of RFP and Tet.

Tomorrow, we can prepare the Master Mix for the digestions of both reporter plamids and then add the restriction enzymes once they arrive. Then, we can follow through with the rest of the plans we had made above in the section titles "Next?".

==Thursday, June 11, 2009==

We began this morning by calculating how many uL we would need for each of the mixes for digesting our reporter plasmids. We decided to double the final volume of each mix to 40uL instead of 20uL. Since we knew the concentration of each plasmid and also knew we needed 2500 ng, we cross multiplied to find the uL of DNA we would need for each.

Concentration Calculations:
310.4 ng/1 uL = 2500ng/x uL
x = 8.05 uL

Here is a chart indicating how much of each we used for the 2 mixes.

[[Image: DoubledigestRFPTetR.png]]

On the Promega website, we found a list of compatible buffers for double digestions. Buffers E and H are compatible for the Tet plasmid and RFP plasmid digestions respectively. We fortunately have both buffers in stock.

[[Image:BamEcoR1.png]] [[Image:NcoEcoR1.png]]

We waited for our waited for the restriction enzymes to arrive and in the meantime, we transformed the plasmids with pLac+RBS, pBad, and pBad+RBS into ''E.coli'' cells. We want the ''E.coli'' cells to replicate the plasmids with these genes because we have a limited amount of genes. [http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_Transformation.html Zippy Plasmid Transformation]

Once our oligos and restriction enzymes arrived, we got to work right away!

'''PCR (Polymerase Chain Reaction)'''

We calculated how much plasmid we will need for the PCR. We decided to dilute the concentration of our plasmids a hundred fold so that we could get pipetable amounts.

''Change in Concentrations''
310.4 ng/uL x 1/100 (dilution factor) = 3.104 ng/uL (RFP)
163.6 ng/uL x 1/100 (dilution factor) = 0.164 ng/uL (Tet)

''Example shown with RFP''
3.104 ng/1 uL = 1ng/ x uL, x = .32
For Tet, x = .61

We also calculated how much of each primer we would need with the following equation:

C1V1 = C2V2
(100 uM) (x) = (1 uM)( 100 uL)
x = 1 uL

[[Image: screen-capture-8.png]]

Each of the 5 of us made had 2 tubes (one for each reporter protein) because there were 5 different forward primers.To each tube, we added the template DNA (either RFP or Tet), water, the respective reverse primer (either RFP or Tet), and the forward primer depending on which 5mer we had chosen. My 5mer was CCAUC. Finally, we added the Master Mix last because this contained the enzyme (DNA polymerase) that would amplify our desired sequence.

[[Image: Pcrtubes.png]]

We followed the temperature cycles so that PCR could occur. The annealing temperature needed to be 5C less than the smallest melting temperature of the primers. Since the reverse primer of RFP had a melting temp of 66C, we set the temperature of the 3rd cycle on 61C.

[[Image: Temperaturecycle.png]]

'''tRNA Oligo Assembly'''

We needed to calculate the concentration of each of the oligo before carrying out the assembly. The stock concentration of each of the oligo was 100 uM. Since we wanted to have 5uM of the oligo in the test tube, and we know that final volume of the test tube is 20 uL, we just cross multiplied to find the amount we needed to add to the tubes to get this concentration.

(100uM)x = (5uM)(20uL)
x = 1 uL

We added 1 uL of each oligo to the tubes.

We had six total tRNAs we needed to assemble so each of us took one FML (one person took two) and to each tube we added 1uL of each oligo for a total of 7uL, 2uL of buffer, and 11uL of water for a total volume of 20 uL. Five of the oligos were constant and 2 varied for each of the different tRNAs. So everyone pipetted the same 5 oligos and everyone also had 2 unique oligos to add. When all of the oligos were added and the tubes ready, we boiled the tubes at 50C for 5 minutes. We then allowed the tubes to remain in the water to cool very slowly overnight so that the annealing process could take place.

The last thing we did at the end of the day was finish off our plasmid digestion that we had begun in the morning. We added the buffer, DNA, water, and restriction enzymes and placed this in the incubator for overnight digestion.

'''To Do''':

1. Isolate our desired dsDNA from the PCR.
2. Verify our tRNA inserts and eventually transform these into plasmids.
3. Check our agar plates to see colonies that have replicated our pLac+RBS, pBAD+RBS, and pBAD genes.

==Friday, June 12, 2009==

This morning, we began by outlining the main items on today's agenda.

1. tRNAs: Ligation, Transformation
2. Digestion reporters (run all of it and gel purify)
3. Transformed cells
4. PCR: Gel run an aliquot to verify (5uL)

We used the equation given by the Ligation protocol to calculate how many nanograms of insert we would need. Initially, we started out calculated the MW average of the 9 and 10 anticodon loops of tRNAs which were 15828.23 ug/umols and 159016.6 ug/umols respectively. We then saw, however, that these would not be helpful values in the process. Instead we started out with the equation below to find the final ng of insert we would need for the ligation.

[[Image: ligationcalculations.png]]

We calculated our initial concentration in ng/uL of insert as shown below.

[[Image: ligationequation.png]]
X nanograms of insert = [(2)(144bp)(50 ng linearalized plasmid)]/2038bp
X = 7.0657 ng of insert

We wanted to find the amount of uL of insert we would need to use. We diluted the initial concentration 50x because we wanted a pipettable amount of insert. Since we knew we wanted the final amount of nanograms to be 7.0657, we calculated as shown below:

''DILUTION'': 555.48/50 = 11.012
''CALCULATION'': 11.012 ng/ 1uL = 7.065 ng/ x uL
x = .64 uL of insert

After we had done these calculations, half of the group started to carryout the ligation of the tRNA inserts into empty plasmids. We needed 7 ligations total: 6 were the tRNAs we needed, while the last one is a negative control. These are the final amounts of what we used:

[[Image: Trnaligation.png]]

The other half of the group (which included me) prepared the agarose gels so that we could run gels for the digested reporters and the PCR inserts. We calculated that the digested reporters needed an agarose of 4% and the PCR reporters needed an agarose of 1.45%. We allowed this gel to form for about 45 minutes to an hour. We prepared both the gels and allowed them to run through lunch.
After lunch, we took pictures of our gels and noticed that the third lane in the PCR gel (third lane from the top in the bottom photo) did not develop a band. The lane was supposed to contain the CUAGU insert for Tetracycline Resistance.

[[Image: 2009-06-12 15hr 54min.jpg]] [[Image:2009-06-12_16hr_07min.jpg]]

Romina Clemente and I decided to repeat this PCR using the TetResistance and RBS+TetResistance plasmids as templates. We labeled J31007 as "2*" and S03710 as "2!". We had to recalculate how much template DNA to use. We let the PCR run overnight. We also verified that the digestion fragments (both the plasmid and the insert) on the other gel were the correct size. The plasmids, which are the brighter fragment in the gel with only 2 lanes, contain the plasmid base pairs along with the rest of our reporter genes. We isolated these plasmids from the gel today and stored them for purification on Monday.

In the meantime, other members of our lab had finished the ligation of the tRNA genes to the empty plasmids and were now transforming these into competent cells. They placed the agar plate with these cells in the fridge overnight.

We also observed the transformations that we had performed yesterday and noticed that I13453 did not form any colonies. J04500 and J3102 did form colonies.

To Do:
1. PCR: Dig, Ligate, Transform
Clean and concentration column, digest, clean and concentration column, ligate.
2. Gel purification of our digestion plasmids
3. Check if colonies have grown for our tRNAs

==Saturday, June 13, 2009==

Alyndria Thompson came in on Saturday morning to remove the tRNA plates and put the PCR reactions in the fridge.

'''tRNA Colonies'''

She counted the colonies of tRNA on each plate.

[[Image:TRNA_colonies_6_13_09.png]]

She placed the plates in the large fridge on the top shelf at 9:23 am. She talked to Dr. Campbell who said we will have to try to "work them up".

'''PCR'''

Products were placed in orange rack of smaller fridge. [2*, 2!]

==Sunday, June 14, 2009==

'''Second Ligation and Transformation of tRNA inserts'''

We decided to re-ligate and re-transform all of the tRNA inserts because we did not get very good yields of colonies from the transformations.
We are using the same measurements for this ligation as we had used on Friday.

[[Image: Trnaligation.png]]

After ligation, we transformed the cells. Using the following measurements, we transferred the competent cells into new tubes.

40uL of competent cells
10uL of ligation mixture
60uL of LB media

After spreading the mixture onto the agar plates, we incubated the plates at 10:56 pm.

We also stored liquid cultures of the colonies that greater than a 2:1 variable:negative control ratio just in case we wanted to use them for mini preps in the future.

IGEM Notebook

2009-06-15T02:46:35Z

ShPunjabi: /* Sunday, June 14, 2009 */

==Wednesday, June 3, 2009==

Romina Clemente and I are trying to find suitable reporter proteins to use. Yesterday, Leland Taylor and Alyndria Thompson were working on ways to insert the gene sequences into the plasmid. Upon seeing how they wanted to manipulated the reporter gene to include the logical clauses, we came up with a few criteria for the reporter genes we would use. The following criteria for genes are listed in order of the broadest aspect to look at to the narrowest aspect:
a) Doesn't contain restriction sites for the 4 restriction enzymes (EcoR1, Xbal, Spel, Pst1) used to cleave the Biobrick part out of the plasmid.
b) Contains 6 cutter restriction sites.
c) These restriction enzymes aren't blunt (cleave straight down at one spot).
d) These restriction sites are close to thge 5' (beginning) end of the sequence.
e) These enzymes are easiest to work with and cheapest.
We are finding the part numbers of the reporter genes we want to use (antibiotic resistance, fluorescence, LacZ) through our own GCAT because we know these ones work. We are then locating these parts on the parts registry [1] website. We copied and pasted the gene sequences we obtained from the registry onto the ApE software [2]. From here, we were able to generate a genetic map of each gene that outlined each restriction site that fit our criteria. We put each genetic map, alongside the part number used, into a Word document.

http://gcat.davidson.edu/GcatWiki/images/0/0e/Restriction_Site_Mapping_on_Reporter_Genes.doc

Later on in the day, we decided that the best way to test for suppression would involve placing the 5mers at the beginning of the reporter gene instead of inserting them into the reporter gene. To do this, we decided that the gene along with its start codon need to be expressed after the 5mer. We will insert a BioBrick plasmid with a 5mer and start codon that will be incorporated before the gene:
RBS-6-8nt-ATG-5mer--ATG-gene

Leland Taylor assembled the oligos that we will need for the BioBrick pieces.

In order to test whether the reporter protein would be expressed regardless of the 5mer being suppressed or not, we would need to remove the suppressor tRNAs from the cell.

Meanwhile, I helped figure out the coding sequence that we will need for the suppressor tRNAs that will bind to the 5mers. We decided that we would be using the following suppressor tRNAs on the Davidson side: CUAGU, CCCUC, CGGUC, CCAUC, and CCACU. We searched through several papers, with little result and finally emailed Dr. Christopher Anderson of UC Berkeley to request the DNA sequence. He emailed us back with a generic DNA code that we could use to create the tRNA; however, for each suppressor tRNA we would use, we would need to change base pairs in the anticodon loop.

The length of the tRNA is 92 nt. The anticodon loop is comprised of 9 nt. Once we have BioBricked the DNA sequence with 4 restriction sites and supplemental nucleotides, the length of the entire gene is 144 nucleotides. This is made up of the 92 nt +22 nt on each side of the sequence (44) + 4 nt on each side of the restriction ends (8).

At the end of the day, we decided to use tetracycline resistance and RFP as our reporter proteins for this tester experiment.

==Thursday, June 4, 2009==

This morning, I began the day by learning how to use the PEARL prgram. Olivia Ho-Shing sat with me and walked me through the program script. And then, Leland Taylor and Shashank Suresh had a problem they wanted us to solve. Leland explained that we need to make sure there is a stop codon in the gene if we fail to suppress the 5mer. The stop codon needs to be before we find another 5mer. They also wanted us to run through both the RFP and Tet Resistance genes to make sure that they did not contain any of the anticodon sequences from our 5 frameshift suppressor tRNAs so as to throw the translation out of frame. They further wanted us to confirm that if we do have successful suppression, there are no stop codons in the middle of the RFP and Tet Resistance genes. PEARL did not find any matches for the suppressor tRNAs within genes. We found 3 matches for stop codons UAA. Two of these occured at the end of the Tet and one at the end of RFP, where they should normally be to end translation into a protein.

Following this, Olivia Ho-Shing and I tried to determine how we can add BioBrick ends to our single stranded DNA sequences that code for the suppressor tRNAs. Our ultimate goal is to find complements for these single strands so they can be put into plasmids. We found the standard prefixes and suffixes for sequences that do not contain ATG (becuase this sequence is a functional RNA and will not be translated) and we placed these before and after the altered 92bp sequence that Dr. Anderson provided us with. The prefixes and suffixes are necessary because they are the extra nucleotides that will be mimicking a "restriction enzyme cutting site". The single strand appeared as below after the Bio Brick prefixes and suffixes were added:

22ntPREFIX--32nt--VARIABLE ANTICODON LOOP--51nt--21ntSUFFIX

We used the suppressor codon CUAGU as our example and placed its appropriate anticodon loop in the sequence. We put this into the lancelator that would give us the other strand as well as the oligos we would need for ideal construction of this double stranded DNA fragment to put into the plasmid. We recieved a total of 4 oligos, 2 of which were variable and 2 of which were constant for all 6 different suppressor tRNAs. Using this double strand, we simulated a restriction enzyme digestion by EcoRI and PstI. We took off the G at both ends of the top strand and then removed the first five nucleotides on each end of the bottom strand. This made the top strand longer than the bottom one. We then proceeded to take the two variable strands and changed the anticodon loop nt for each suppressor tRNA we used.

Around lunchtime, we encountered a problem with the BioBrick scar in the "ATG-5mer subpiece" of the test project. It turns out that after annealing the sticky ends of the Xba1 and Spe1 sites, the scar created had a stop codon TAG (UAG) that was in frame with the suppressed codon. When the Xba1 site on insert anneals to the Spe1 site on the plasmid (the green portions), we get a scar that reads TACTAG.
[[Image:screen-capture.png]]

For the afternoon, Olivia Ho-Shing and I finished assembly of the oligos of the suppressor tRNA codes. We assembled the document below:

http://gcat.davidson.edu/GcatWiki/images/5/5e/TRNAoligostoOrder.doc

We were assigned tasks of creating "cartoons" to illustrate the phenomenon that had occurred. No one could successfully do this however, because we could not understand how the piece had formed. The parts registry website provided wrong information on the restriction site of Spe1 which slowed our efficiency. This project turned into an effort to try to find two other different restriction enzymes that have complementary sticky ends other than Xba1 and Spe1. We want to get rid oc the scar that contains TACTAG.

We ended up deciding that a hybrid of two ideas was needed: restriction enzymes would need to be used to make several reporter gene sequences with different beginnings dependending on the suppressor tRNA sequence needed.

==Friday, June 5, 2009==

Our goal for this morning was to discuss the hybrid PCR approach to assemble the part that comes before the gene we wanted to express. We decided that we would have a BioBrick prefix and a 5mer followed by a sequence of nucleotides whose identity was up to us. Using PCR we would get the second double strand if we have the following:

BioBrick Prefix--ATG--5mer--gene until we get to restriction site

The PCR primer would attach to the ATG and the 5mer and would replicate the complementary base pairs to make a double stranded DNA sequence. As several of us were confused about PCR methods, we looked at the online link provided by Alyndria Thompson's link to the PCR interactive website which was very helpful. We also did some research to discover that the restriction enzyme BseRI (which is contained in the YFP that we wanted to alter) is not compatible with any other restriction enzyme. This is good because we did not want to plasmid containing this gene to close up with Xba1 once we digested it.

The team experienced some confusion with the hybrid idea and spoke to Clif Davis of Missouri Western University on the phone. He told us that there will be one part that is a fragment of the beginning of some nucleotides as a restriction cleaving site, ATG, the logical clause, and the start of the YFP gene that will be placed into a plasmid containing a space. To the left of the space will be the BioBrick Prefix and the right of the space will contain the rest of the genes in the YFP. Therefore, we decided not to alter any BioBrick ends and just look at restriction sites at the beginning of reporter proteins. The small fragment that will be placed into the plasmid will be made by PCR. The PCR will attach a primer onto the ATG and 5mer and replicate.

While we were discussing this, we expanded upon the idea that we can do whatever we want with the "suffix" portion between the 5mer and the beginning of YFP. We decided it would be best to remove ATG and add the first 5 amino acids and the restriction enzyme site. Along with this, if we have the 5mer coding for a certain amino acid (ie methionine) we could replace another codon that codes for methionine WITH that 5mer so long as this codon is contained within the first 15 nt. This way, we would not be altering the protein.

Olivia Ho-Shing came up with an idea of cascading PCR effect for longer logical clauses. Post translational modification of the protein was another one of her ideas. This applies because although we may code for the protein in a given way, the protein may modify or fix itself up to perform a slightly different function in its tertiary form.

Dr. Campbell suggested that we can look up restriction enzymes 400-500 base pairs into the reporter protein we would like to use; however, we have more flexibility in our procedure if we choose more "hardy" cutters. So instead, we could cut with EcoRI instead of with Xba1.

During the afternoon, we set about three tasks:
1) Which primers to use
2) Which promoters to use
3) Which restriction enzymes and how they will determine use of reporter proteins

Olivia Ho-Shing and I began looking up which restriction enzymes were strong so we could base our reporters on that. We began with Tet and RFP since those were the two reporters we had started out with choosing. I found a website that provided enzyme cleavage efficiency based upon the proximity of the site to the end of the PCR fragment that contains it. We discovered the following based upon the attached documents:

http://gcat.davidson.edu/GcatWiki/images/8/8f/Restriction_Enzyme_Cleavage_Efficiency.doc
http://gcat.davidson.edu/GcatWiki/images/9/9a/07Jun09_RE_Cutting_Efficiency_oh.doc

We decided that these two reporters contained efficient enzymes in a good quantity to choose from. On Monday, we will look through these enzymes once more and finalize which enzymes we would like to use with RFP and Tet.

==Sunday, June 7, 2009==

We did preparations for the mini prep this evening. The following notes are courtesy of Olivia Ho-Shing.

1. Sterically transfer ~2mL LB+AMP (AMP are ampicillin resistant plasmids) *Never put fluid back into bottle once it has entered pipette*
2. Use forceps to grab toothpick, scrap some frozen cells, rub in tube with top

'''Tubes'''

S03511 (2)

K091111

B0030

I715039

K091112

I715039

J31007 (2)

B0034

E1010

S03710

==Monday, June 8, 2009==

We reviewed the PCR process this morning as well as the restriction enzyme cleavage process.

Then, Leland Taylor, Olivia Ho-Shing and I all worked on converting the tRNA oligos we made to the template that Missouri Western used. The oligos we had previously made were different from the ones that Dr. Anderson had originally emailed us because further communication between Dr. Anderson and Missouri Western resulted in a better template.

We tried several times to minimize the number of oligos while keeping their individual lengths under 80bp. We managed to do this in the end by creating 7 oligos, 2 of which are variable between the suppressor tRNAs.
We reviewed the following document and finalized the oligos that we needed to order for the frameshift suppressor tRNAs. We decided to keep both the 10bp and 9bp anticodons of CCAUC.

I also looked up the amino acids that corresponded to the FS (frameshift suppressor) tRNAs for future use:

1. CUAGU = Leu
2. CCCUC = Pro
3. CGGUC = Arg
4. CCACU = Pro
5. CCAUC* = Pro
*9 and 10 bp anticodons

Also, we have decided to use NcoI as the restriction enzyme for RFP. This cleavage site occurs 419 bp into the gene. We have decided to use BamHI as the enzyme for Tetracycline Resistance and the cleavage site occurs 290bp into the gene. Both of these enzymes are very powerful and hardy.

We also looked over Missouri Western's tRNA oligos and primer oligos which we approved.
Our oligos were also approved by Missouri Western and Dr. Campbell ordered them while we supervised.

In the afternoon we did the miniprep. The mini prep directions were as appear in the link below.

http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_MiniPrep.html

We then worked with the Nano Prep to dilute our solution of DNA to an absorption of 1.5 and then we made labels for the tubes. The chart below shows relevant information we obtained from the NanoDrop for each of the reporter plasmids.

[[Image: Screen-capture-6.png]]

==Tuesday, June 9, 2009==

'''Preparations for restriction enzymes digestion of plasmids'''

Purpose: To determine the size of the plasmid inserts and ensure that plasmids are what we extracted from the cells during the mini-prep.

Things we need:
DNA ~3uL
Buffer 10x (Want to dilute to 1x) so add 2uL
Enzyme 1 ~1uL
Enzme 2 ~1uL
Water ~13uL
Final Volunter about 20 uL.

Need to choose the right buffer for the restriction enzymes. All buffers come in 10x (10 times more concentrated than what you want it to be when you use it). [http://gcat.davidson.edu/GcatWiki/index.php/Davidson_Missouri_W/Double_Digest_Guide Buffer Website]

Total volume of enzymes has to be 10% or less than final volume (most volume 2uL). Barely submerge the pipet tip into the solution when pipeting the enzymes.
Star (*) activity: Restriction enzyme stops cutting specifically and starts cutting everywhere.

DNA can't be seen by itself so we add EtBr (the bigger the insert, the more will bind) so bigger strands appear brighter. samples.

The order of entry doesn't matter, but make sure to enter the enzymes last.

Will have lots of labeled tubes and will put 3uL of appropriate plasmids into the tubes.
In the meantime, we'll make a "Master Mix" of all the other ingredients in a bigger tube. Always make enough volume for one extra tube.

Enzymes usually have an optimal temperature of 37C. We will let this reaction go for an hour and then we will run it on a gel.

'''Gel Electrophoresis Protocol'''

Website online for ideal gel concentration for resolving different sizes of molecules [http://gcat.davidson.edu/iGEM08/gelwebsite/gelwebsite.html Gel Concentration Website].
See protocol on the Davidson/Missouri Western Wookie for running molecules in the gel and how to digest with restriction enzymes.

'''What's Next?'''

1. 5' end additions
a. Using PCR
b. Cut at the restriction sites EcoR1 and either Bam H1 or Nco1
c. Cut plasmid and reporter with same restriction enzymes as above
d. Run a gel and purify "keeper" DNA (Ligate transform, screen, sequence)
e. Then, we take the "keeper" DNA and combine it with the insert from part

We can have steps d and e ready in advance.

2. tRNAs
a. Assemble the oligos into genes (Requires lots of calculations beforehand, but the actual steps )
b. Plasmids cut up with EcoR1 and Pst1 '''WE CAN USE THE "WASTE" PLASMID FROM THE RESTRICTION ENZYME DIGEST'''
c. Gel purify the plasmids (Ligate transform, screen, sequence)

The following tables include Master Mix measurements, agarose gel concentrations and buffers.
http://gcat.davidson.edu/GcatWiki/images/0/08/Shamitapic3.png
http://gcat.davidson.edu/GcatWiki/images/d/d1/Shamitapic4.png
http://gcat.davidson.edu/GcatWiki/images/6/6f/Shamitapic5.png

We made the Master Mix with our DNA and put it in the incubator at 10:40 AM.

'''Preparing the Gels'''

Now, we will be making 2 gels, 1 at 0.8% and 2.5% (weight to volume percent). The gel volume is 60mL.

'''Definition of 1% solution:
1 gram of agarose/ 100mL buffer'''

So for a 0.8% solution: 0.8 grams agarose/100mL buffer AND we need 60mL (fixed volume for casts run) so multiply that by 0.6.
Final concentration:

.48 grams agarose/60mL buffer

1 uL of EtBr stock for 60mL of gel.
EtBr is in the buffer so it is constantly replenishing itself even though it is moving toward one end in order to make bands on the other end visible.

After Dr. Campbell's demonstration, we each helped prepare a 0.8% buffer solution using the agarose powder, buffer and EtBr. We mixed together the first two substances in a 200mL Erlenmeyer flask and heated it for 120 seconds in the microwave. We then added EtBr and poured the gel solution into the gel cast.

'''Agar Plate'''

After lunch, we grew some more cells for each of the 11 parts in an agar plate labeled "LBAMP-AMC-9 June" so that so would have extra cells that could be stored for up to 2 weeks.
We made small circles on the agar plate and numbered them 1-11. The numbers corresponded to the following parts. We put the plate in the incubator overnight to let the cells grow.

1. B0030 AMC
2. K091111
3. S03710
4. I715039 OEH
5. S03511 ORH
6. I715039 AST
7. B0030 SDP
8. K091112
9. J31007 LJH
10. J31007 REC
11. S03511

'''Unloading the Gels'''

The following shows which parts we entered in which gels:

[[Image: screen-capture-2.png]]

We unloaded the 2.5% agarose first and after observing the bands under a UV light, put the gel into the BioRad machine to observe the bands. The image below is of a Molecular Weight Marker that shows the number of bp in control fragments of DNA according to the distance they travel down the gel.

[[Image: DNA_molecular_weight_marker_gel.png]]

We obtained the following printout for the first gel that was 2.5% Agarose.
[[Image: 2009-06-09_14hr_44min.jpg]]

The bands in the first well at the bottom are those of the molecular weight marker. We skipped the lane above (to the right) of the MWM and entered the rest of the inserts as described in the above table. A light band is visible for each of the 4 other wells. In Lanes 3 and 4 we concluded we are supposed to have approximately 45 bp because the insert is 15bp + 30 bp on either side of the insert (Xba1 and Spe1 restrictions sites + sticky ends + extra bases). We used the same logic for Lanes 5 and 6 which are supposed to contain approximately 86 base pairs.

One discrepancy in this gel, however, is that Lanes 5 and 6, which according to their labels respectively contain the LacIQ promoter and the Lac IQ1 promoter. According to the information online at the parts registry website, pLacIQ1 should be the same length as pLacIQ. The gels do not show this, but instead show that the sample in Lane 5 is smaller than that in Lane 6 because Lane 5 traveled further. Leland Taylor and Romina Clemente sent out an email to Pallavi Penumetcha to ask about this because she had written a paper about this.

We then unloaded the 0.8% agarose gel and then placed this in the BioRad machine under the same protocol as above. The following image is the 0.8% Agarose.

[[Image:2009-06-09_15hr_21min.jpg]]

We observed that the bands in Lane 1 (which is now at the top) are examples of high intensity bands. The plasmids contained in the original well are twice the intensity of the insert.

In Lane 5, however, we observed several different bands. This could be due to the extra DNA that was sitting on the top of the well. We decided to re-expose the gel to get better observations on Lanes 3 and 4 because the bands were not as intense. We anticipated seeing a band at about 250 bp because this was the size of the insert (plus extra bp). After re-exposing the gel, we did not find any difference and still did not see any bands in these lanes. We have decided to redo the gel on the part S03511 and so we prepared the master mix with that DNA and incubated it so that we could run the gel tomorrow.

We also cut out the "empty" plasmids from the gel so that we could use this as vectors for the coding sequence of the suppressor tRNAs.

Tomorrow we will be planning out specific tasks for people once the oligos arrive.

==Wednesday, June 10, 2009==

We began this morning by making the agarose gel with the same protocol as yesterday. This time, since we are just re-doing both S03511 inserts, we will have a 1.8% agarose gel.

'''OUTLINE: WHERE DO WE STAND?'''

Yesterday
a. Isolated empty plasmids
b. Verified most inserts
c. Waiting for:
>tRNA oligos
>Primers
a. Will amplify the reporter gene in the plasmid itself

Next?
a. When Primers Arrive
> Amplify the reporter genes with primers (PCR takes ~3 hours)
> Calculate how much reporter plasmid we will need (Start with 50x more than we actually will need)
> Cut amplified sequence (EcoR1 and BamH1/Nco1) then purify what we need
> Cut reporter plasmid (EcoR1 and BamH1/Nco1) then purify what we need
> Ligate amplified sequence and reporter plasmid
b. When tRNA oligos arrive
> Calculate how much of each oligo we will need (takes a while...)
> "Boil and cool" tRNA oligos together
> Ligate fragments (dsDNA) into plasmids we isolated yesterday (~5 minutes)
> Transform the plasmids (~20 minutes)

* LOOK THROUGH LIGATION PROTOCOL (How to assemble genes)
* LOOK THROUGH TRANSFORMATION PROTOCOL

''What we need to get done today'':
1. Prepare the control for the Tet Resistance by BioBricking the part pLac+RBS (S03511) with the TetA (J31007).
2. Check the part number for RFP and also for pLac+RBS+RFP
3. Read through ligation protocol
4. See how many nanograms of plasmid we will need for the tRNAs and for the inserts and multiply this number by 50.
> Check if we have enough plasmids from our mini-prep
5. Purify the plasmids from the gel

After we made this outline, Leland, Olivia and I analyzed the gel we ran this morning and did not see a more than one band in the 2 lanes that contained DNA from S03511 (image below). Since we did not see multiple bands in yesterday's gel either, we conclude that there is something wrong with the DNA rather than with the restriction enzymes that we used to cut it. We concluded that there is probably not an insert in this plasmid both the part and the plasmid are useless.

[[Image: 2009-06-10 10hr 30min.jpg]]

Now, we do not have an immediately accessible promoter. We have decided to order the pBAD promoter from Missouri Western and we will attach the RBS+Tet to this plasmid. Our insert for this will be the larger gene because it is easier to manipulate in the gel than the small gene.

Alyndria Thompson and Romina Clemente are figuring out the size of the plasmids including the reporter genes.

After lunch we performed several steps to isolate and purify the gel to get the empty plasmids will be the vectors for the tRNA DNA. Then, we used the NanoDrop to assess the purity of our DNA. We found that the plasmids were more pure than we had expected. The following values were obtained:

Abs 1.850
A-260 10 mm path 0.172
A-280 10 mm path 0.075
260/280 2.28
260/230 0.09

We then followed the protocol for preparing the agar plates. We put the plate with the agar solution in the Autoclave.

While the solution was in the autoclave, we discussed that we don't really need a promoter in front of RBS+Tet for the gene to be expressed because Tet expression is so strong that the gene would translate without a promoter. However, it would be a good idea to have the promoter because if it was possible to reinforce the translation of the reporter protein, we wanted to do that.

We found that there were some tubes in the lab fridge that contained the pBAD and pLac promoters with RBS. We decided to use these as the promoter and RBS for our experiments. The pLac promoter will be used for RFP and the pBad promoter used with Tet Resistance. We decided to use the pBad with Tet because it is weaker in comparison to pLac. As expression of the Tet gene can be toxic to a cell, we do not want to overexpress the gene with a stronger promoter.

Once the agar solution was sterilized in the Autoclave, we removed it, cooled it, and pipetted the antibiotic resistance into it. We then poured the solution into the plates, removed any bubbles, and let the gel form.

Alyndria Thompson and Romina Clemente concluded that we had more than enough recieving vectors for both the RFP and Tetracycline genes. Since we need 50 nanograms, it is wise to have at least 50 times that much that we can work with, so we need at least 2,500 ng.

RFP 1: 77.6 x 4 = 310.4 ng/uL
x 20 uL = 3,272 ng
RFP 2: 59.5 x 4 = 238 ng/uL
x 20 uL = 4,760 ng
Tet: 40.9 x 4 = 163.6 ng/uL
x 20 uL = 3,272 ng

According to these calculations we have more than 50 times what we need for both instances of RFP and Tet.

Tomorrow, we can prepare the Master Mix for the digestions of both reporter plamids and then add the restriction enzymes once they arrive. Then, we can follow through with the rest of the plans we had made above in the section titles "Next?".

==Thursday, June 11, 2009==

We began this morning by calculating how many uL we would need for each of the mixes for digesting our reporter plasmids. We decided to double the final volume of each mix to 40uL instead of 20uL. Since we knew the concentration of each plasmid and also knew we needed 2500 ng, we cross multiplied to find the uL of DNA we would need for each.

Concentration Calculations:
310.4 ng/1 uL = 2500ng/x uL
x = 8.05 uL

Here is a chart indicating how much of each we used for the 2 mixes.

[[Image: DoubledigestRFPTetR.png]]

On the Promega website, we found a list of compatible buffers for double digestions. Buffers E and H are compatible for the Tet plasmid and RFP plasmid digestions respectively. We fortunately have both buffers in stock.

[[Image:BamEcoR1.png]] [[Image:NcoEcoR1.png]]

We waited for our waited for the restriction enzymes to arrive and in the meantime, we transformed the plasmids with pLac+RBS, pBad, and pBad+RBS into ''E.coli'' cells. We want the ''E.coli'' cells to replicate the plasmids with these genes because we have a limited amount of genes. [http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_Transformation.html Zippy Plasmid Transformation]

Once our oligos and restriction enzymes arrived, we got to work right away!

'''PCR (Polymerase Chain Reaction)'''

We calculated how much plasmid we will need for the PCR. We decided to dilute the concentration of our plasmids a hundred fold so that we could get pipetable amounts.

''Change in Concentrations''
310.4 ng/uL x 1/100 (dilution factor) = 3.104 ng/uL (RFP)
163.6 ng/uL x 1/100 (dilution factor) = 0.164 ng/uL (Tet)

''Example shown with RFP''
3.104 ng/1 uL = 1ng/ x uL, x = .32
For Tet, x = .61

We also calculated how much of each primer we would need with the following equation:

C1V1 = C2V2
(100 uM) (x) = (1 uM)( 100 uL)
x = 1 uL

[[Image: screen-capture-8.png]]

Each of the 5 of us made had 2 tubes (one for each reporter protein) because there were 5 different forward primers.To each tube, we added the template DNA (either RFP or Tet), water, the respective reverse primer (either RFP or Tet), and the forward primer depending on which 5mer we had chosen. My 5mer was CCAUC. Finally, we added the Master Mix last because this contained the enzyme (DNA polymerase) that would amplify our desired sequence.

[[Image: Pcrtubes.png]]

We followed the temperature cycles so that PCR could occur. The annealing temperature needed to be 5C less than the smallest melting temperature of the primers. Since the reverse primer of RFP had a melting temp of 66C, we set the temperature of the 3rd cycle on 61C.

[[Image: Temperaturecycle.png]]

'''tRNA Oligo Assembly'''

We needed to calculate the concentration of each of the oligo before carrying out the assembly. The stock concentration of each of the oligo was 100 uM. Since we wanted to have 5uM of the oligo in the test tube, and we know that final volume of the test tube is 20 uL, we just cross multiplied to find the amount we needed to add to the tubes to get this concentration.

(100uM)x = (5uM)(20uL)
x = 1 uL

We added 1 uL of each oligo to the tubes.

We had six total tRNAs we needed to assemble so each of us took one FML (one person took two) and to each tube we added 1uL of each oligo for a total of 7uL, 2uL of buffer, and 11uL of water for a total volume of 20 uL. Five of the oligos were constant and 2 varied for each of the different tRNAs. So everyone pipetted the same 5 oligos and everyone also had 2 unique oligos to add. When all of the oligos were added and the tubes ready, we boiled the tubes at 50C for 5 minutes. We then allowed the tubes to remain in the water to cool very slowly overnight so that the annealing process could take place.

The last thing we did at the end of the day was finish off our plasmid digestion that we had begun in the morning. We added the buffer, DNA, water, and restriction enzymes and placed this in the incubator for overnight digestion.

'''To Do''':

1. Isolate our desired dsDNA from the PCR.
2. Verify our tRNA inserts and eventually transform these into plasmids.
3. Check our agar plates to see colonies that have replicated our pLac+RBS, pBAD+RBS, and pBAD genes.

==Friday, June 12, 2009==

This morning, we began by outlining the main items on today's agenda.

1. tRNAs: Ligation, Transformation
2. Digestion reporters (run all of it and gel purify)
3. Transformed cells
4. PCR: Gel run an aliquot to verify (5uL)

We used the equation given by the Ligation protocol to calculate how many nanograms of insert we would need. Initially, we started out calculated the MW average of the 9 and 10 anticodon loops of tRNAs which were 15828.23 ug/umols and 159016.6 ug/umols respectively. We then saw, however, that these would not be helpful values in the process. Instead we started out with the equation below to find the final ng of insert we would need for the ligation.

[[Image: ligationcalculations.png]]

We calculated our initial concentration in ng/uL of insert as shown below.

[[Image: ligationequation.png]]
X nanograms of insert = [(2)(144bp)(50 ng linearalized plasmid)]/2038bp
X = 7.0657 ng of insert

We wanted to find the amount of uL of insert we would need to use. We diluted the initial concentration 50x because we wanted a pipettable amount of insert. Since we knew we wanted the final amount of nanograms to be 7.0657, we calculated as shown below:

''DILUTION'': 555.48/50 = 11.012
''CALCULATION'': 11.012 ng/ 1uL = 7.065 ng/ x uL
x = .64 uL of insert

After we had done these calculations, half of the group started to carryout the ligation of the tRNA inserts into empty plasmids. We needed 7 ligations total: 6 were the tRNAs we needed, while the last one is a negative control. These are the final amounts of what we used:

[[Image: Trnaligation.png]]

The other half of the group (which included me) prepared the agarose gels so that we could run gels for the digested reporters and the PCR inserts. We calculated that the digested reporters needed an agarose of 4% and the PCR reporters needed an agarose of 1.45%. We allowed this gel to form for about 45 minutes to an hour. We prepared both the gels and allowed them to run through lunch.
After lunch, we took pictures of our gels and noticed that the third lane in the PCR gel (third lane from the top in the bottom photo) did not develop a band. The lane was supposed to contain the CUAGU insert for Tetracycline Resistance.

[[Image: 2009-06-12 15hr 54min.jpg]] [[Image:2009-06-12_16hr_07min.jpg]]

Romina Clemente and I decided to repeat this PCR using the TetResistance and RBS+TetResistance plasmids as templates. We labeled J31007 as "2*" and S03710 as "2!". We had to recalculate how much template DNA to use. We let the PCR run overnight. We also verified that the digestion fragments (both the plasmid and the insert) on the other gel were the correct size. The plasmids, which are the brighter fragment in the gel with only 2 lanes, contain the plasmid base pairs along with the rest of our reporter genes. We isolated these plasmids from the gel today and stored them for purification on Monday.

In the meantime, other members of our lab had finished the ligation of the tRNA genes to the empty plasmids and were now transforming these into competent cells. They placed the agar plate with these cells in the fridge overnight.

We also observed the transformations that we had performed yesterday and noticed that I13453 did not form any colonies. J04500 and J3102 did form colonies.

To Do:
1. PCR: Dig, Ligate, Transform
Clean and concentration column, digest, clean and concentration column, ligate.
2. Gel purification of our digestion plasmids
3. Check if colonies have grown for our tRNAs

==Saturday, June 13, 2009==

Alyndria Thompson came in on Saturday morning to remove the tRNA plates and put the PCR reactions in the fridge.

'''tRNA Colonies'''

She counted the colonies of tRNA on each plate.

[[Image:TRNA_colonies_6_13_09.png]]

She placed the plates in the large fridge on the top shelf at 9:23 am. She talked to Dr. Campbell who said we will have to try to "work them up".

'''PCR'''

Products were placed in orange rack of smaller fridge. [2*, 2!]

==Sunday, June 14, 2009==

'''Second Ligation and Transformation of tRNA inserts'''

We decided to re-ligate and re-transform all of the tRNA inserts because we did not get very good yields of colonies from the transformations.
We are using the same measurements for this ligation as we had used on Friday.

[[Image: Trnaligation.png]]

After ligation, we transformed the cells. Using the following measurements, we transferred the competent cells into new tubes.

40uL of competent cells
10uL of ligation mixture
60uL of LB media

We also stored liquid cultures of the colonies that greater than a 2:1 variable:negative control ratio just in case we wanted to use them for mini preps in the future.

IGEM Notebook

2009-06-15T02:16:22Z

ShPunjabi: /* Sunday, June 14, 2009 */

==Wednesday, June 3, 2009==

Romina Clemente and I are trying to find suitable reporter proteins to use. Yesterday, Leland Taylor and Alyndria Thompson were working on ways to insert the gene sequences into the plasmid. Upon seeing how they wanted to manipulated the reporter gene to include the logical clauses, we came up with a few criteria for the reporter genes we would use. The following criteria for genes are listed in order of the broadest aspect to look at to the narrowest aspect:
a) Doesn't contain restriction sites for the 4 restriction enzymes (EcoR1, Xbal, Spel, Pst1) used to cleave the Biobrick part out of the plasmid.
b) Contains 6 cutter restriction sites.
c) These restriction enzymes aren't blunt (cleave straight down at one spot).
d) These restriction sites are close to thge 5' (beginning) end of the sequence.
e) These enzymes are easiest to work with and cheapest.
We are finding the part numbers of the reporter genes we want to use (antibiotic resistance, fluorescence, LacZ) through our own GCAT because we know these ones work. We are then locating these parts on the parts registry [1] website. We copied and pasted the gene sequences we obtained from the registry onto the ApE software [2]. From here, we were able to generate a genetic map of each gene that outlined each restriction site that fit our criteria. We put each genetic map, alongside the part number used, into a Word document.

http://gcat.davidson.edu/GcatWiki/images/0/0e/Restriction_Site_Mapping_on_Reporter_Genes.doc

Later on in the day, we decided that the best way to test for suppression would involve placing the 5mers at the beginning of the reporter gene instead of inserting them into the reporter gene. To do this, we decided that the gene along with its start codon need to be expressed after the 5mer. We will insert a BioBrick plasmid with a 5mer and start codon that will be incorporated before the gene:
RBS-6-8nt-ATG-5mer--ATG-gene

Leland Taylor assembled the oligos that we will need for the BioBrick pieces.

In order to test whether the reporter protein would be expressed regardless of the 5mer being suppressed or not, we would need to remove the suppressor tRNAs from the cell.

Meanwhile, I helped figure out the coding sequence that we will need for the suppressor tRNAs that will bind to the 5mers. We decided that we would be using the following suppressor tRNAs on the Davidson side: CUAGU, CCCUC, CGGUC, CCAUC, and CCACU. We searched through several papers, with little result and finally emailed Dr. Christopher Anderson of UC Berkeley to request the DNA sequence. He emailed us back with a generic DNA code that we could use to create the tRNA; however, for each suppressor tRNA we would use, we would need to change base pairs in the anticodon loop.

The length of the tRNA is 92 nt. The anticodon loop is comprised of 9 nt. Once we have BioBricked the DNA sequence with 4 restriction sites and supplemental nucleotides, the length of the entire gene is 144 nucleotides. This is made up of the 92 nt +22 nt on each side of the sequence (44) + 4 nt on each side of the restriction ends (8).

At the end of the day, we decided to use tetracycline resistance and RFP as our reporter proteins for this tester experiment.

==Thursday, June 4, 2009==

This morning, I began the day by learning how to use the PEARL prgram. Olivia Ho-Shing sat with me and walked me through the program script. And then, Leland Taylor and Shashank Suresh had a problem they wanted us to solve. Leland explained that we need to make sure there is a stop codon in the gene if we fail to suppress the 5mer. The stop codon needs to be before we find another 5mer. They also wanted us to run through both the RFP and Tet Resistance genes to make sure that they did not contain any of the anticodon sequences from our 5 frameshift suppressor tRNAs so as to throw the translation out of frame. They further wanted us to confirm that if we do have successful suppression, there are no stop codons in the middle of the RFP and Tet Resistance genes. PEARL did not find any matches for the suppressor tRNAs within genes. We found 3 matches for stop codons UAA. Two of these occured at the end of the Tet and one at the end of RFP, where they should normally be to end translation into a protein.

Following this, Olivia Ho-Shing and I tried to determine how we can add BioBrick ends to our single stranded DNA sequences that code for the suppressor tRNAs. Our ultimate goal is to find complements for these single strands so they can be put into plasmids. We found the standard prefixes and suffixes for sequences that do not contain ATG (becuase this sequence is a functional RNA and will not be translated) and we placed these before and after the altered 92bp sequence that Dr. Anderson provided us with. The prefixes and suffixes are necessary because they are the extra nucleotides that will be mimicking a "restriction enzyme cutting site". The single strand appeared as below after the Bio Brick prefixes and suffixes were added:

22ntPREFIX--32nt--VARIABLE ANTICODON LOOP--51nt--21ntSUFFIX

We used the suppressor codon CUAGU as our example and placed its appropriate anticodon loop in the sequence. We put this into the lancelator that would give us the other strand as well as the oligos we would need for ideal construction of this double stranded DNA fragment to put into the plasmid. We recieved a total of 4 oligos, 2 of which were variable and 2 of which were constant for all 6 different suppressor tRNAs. Using this double strand, we simulated a restriction enzyme digestion by EcoRI and PstI. We took off the G at both ends of the top strand and then removed the first five nucleotides on each end of the bottom strand. This made the top strand longer than the bottom one. We then proceeded to take the two variable strands and changed the anticodon loop nt for each suppressor tRNA we used.

Around lunchtime, we encountered a problem with the BioBrick scar in the "ATG-5mer subpiece" of the test project. It turns out that after annealing the sticky ends of the Xba1 and Spe1 sites, the scar created had a stop codon TAG (UAG) that was in frame with the suppressed codon. When the Xba1 site on insert anneals to the Spe1 site on the plasmid (the green portions), we get a scar that reads TACTAG.
[[Image:screen-capture.png]]

For the afternoon, Olivia Ho-Shing and I finished assembly of the oligos of the suppressor tRNA codes. We assembled the document below:

http://gcat.davidson.edu/GcatWiki/images/5/5e/TRNAoligostoOrder.doc

We were assigned tasks of creating "cartoons" to illustrate the phenomenon that had occurred. No one could successfully do this however, because we could not understand how the piece had formed. The parts registry website provided wrong information on the restriction site of Spe1 which slowed our efficiency. This project turned into an effort to try to find two other different restriction enzymes that have complementary sticky ends other than Xba1 and Spe1. We want to get rid oc the scar that contains TACTAG.

We ended up deciding that a hybrid of two ideas was needed: restriction enzymes would need to be used to make several reporter gene sequences with different beginnings dependending on the suppressor tRNA sequence needed.

==Friday, June 5, 2009==

Our goal for this morning was to discuss the hybrid PCR approach to assemble the part that comes before the gene we wanted to express. We decided that we would have a BioBrick prefix and a 5mer followed by a sequence of nucleotides whose identity was up to us. Using PCR we would get the second double strand if we have the following:

BioBrick Prefix--ATG--5mer--gene until we get to restriction site

The PCR primer would attach to the ATG and the 5mer and would replicate the complementary base pairs to make a double stranded DNA sequence. As several of us were confused about PCR methods, we looked at the online link provided by Alyndria Thompson's link to the PCR interactive website which was very helpful. We also did some research to discover that the restriction enzyme BseRI (which is contained in the YFP that we wanted to alter) is not compatible with any other restriction enzyme. This is good because we did not want to plasmid containing this gene to close up with Xba1 once we digested it.

The team experienced some confusion with the hybrid idea and spoke to Clif Davis of Missouri Western University on the phone. He told us that there will be one part that is a fragment of the beginning of some nucleotides as a restriction cleaving site, ATG, the logical clause, and the start of the YFP gene that will be placed into a plasmid containing a space. To the left of the space will be the BioBrick Prefix and the right of the space will contain the rest of the genes in the YFP. Therefore, we decided not to alter any BioBrick ends and just look at restriction sites at the beginning of reporter proteins. The small fragment that will be placed into the plasmid will be made by PCR. The PCR will attach a primer onto the ATG and 5mer and replicate.

While we were discussing this, we expanded upon the idea that we can do whatever we want with the "suffix" portion between the 5mer and the beginning of YFP. We decided it would be best to remove ATG and add the first 5 amino acids and the restriction enzyme site. Along with this, if we have the 5mer coding for a certain amino acid (ie methionine) we could replace another codon that codes for methionine WITH that 5mer so long as this codon is contained within the first 15 nt. This way, we would not be altering the protein.

Olivia Ho-Shing came up with an idea of cascading PCR effect for longer logical clauses. Post translational modification of the protein was another one of her ideas. This applies because although we may code for the protein in a given way, the protein may modify or fix itself up to perform a slightly different function in its tertiary form.

Dr. Campbell suggested that we can look up restriction enzymes 400-500 base pairs into the reporter protein we would like to use; however, we have more flexibility in our procedure if we choose more "hardy" cutters. So instead, we could cut with EcoRI instead of with Xba1.

During the afternoon, we set about three tasks:
1) Which primers to use
2) Which promoters to use
3) Which restriction enzymes and how they will determine use of reporter proteins

Olivia Ho-Shing and I began looking up which restriction enzymes were strong so we could base our reporters on that. We began with Tet and RFP since those were the two reporters we had started out with choosing. I found a website that provided enzyme cleavage efficiency based upon the proximity of the site to the end of the PCR fragment that contains it. We discovered the following based upon the attached documents:

http://gcat.davidson.edu/GcatWiki/images/8/8f/Restriction_Enzyme_Cleavage_Efficiency.doc
http://gcat.davidson.edu/GcatWiki/images/9/9a/07Jun09_RE_Cutting_Efficiency_oh.doc

We decided that these two reporters contained efficient enzymes in a good quantity to choose from. On Monday, we will look through these enzymes once more and finalize which enzymes we would like to use with RFP and Tet.

==Sunday, June 7, 2009==

We did preparations for the mini prep this evening. The following notes are courtesy of Olivia Ho-Shing.

1. Sterically transfer ~2mL LB+AMP (AMP are ampicillin resistant plasmids) *Never put fluid back into bottle once it has entered pipette*
2. Use forceps to grab toothpick, scrap some frozen cells, rub in tube with top

'''Tubes'''

S03511 (2)

K091111

B0030

I715039

K091112

I715039

J31007 (2)

B0034

E1010

S03710

==Monday, June 8, 2009==

We reviewed the PCR process this morning as well as the restriction enzyme cleavage process.

Then, Leland Taylor, Olivia Ho-Shing and I all worked on converting the tRNA oligos we made to the template that Missouri Western used. The oligos we had previously made were different from the ones that Dr. Anderson had originally emailed us because further communication between Dr. Anderson and Missouri Western resulted in a better template.

We tried several times to minimize the number of oligos while keeping their individual lengths under 80bp. We managed to do this in the end by creating 7 oligos, 2 of which are variable between the suppressor tRNAs.
We reviewed the following document and finalized the oligos that we needed to order for the frameshift suppressor tRNAs. We decided to keep both the 10bp and 9bp anticodons of CCAUC.

I also looked up the amino acids that corresponded to the FS (frameshift suppressor) tRNAs for future use:

1. CUAGU = Leu
2. CCCUC = Pro
3. CGGUC = Arg
4. CCACU = Pro
5. CCAUC* = Pro
*9 and 10 bp anticodons

Also, we have decided to use NcoI as the restriction enzyme for RFP. This cleavage site occurs 419 bp into the gene. We have decided to use BamHI as the enzyme for Tetracycline Resistance and the cleavage site occurs 290bp into the gene. Both of these enzymes are very powerful and hardy.

We also looked over Missouri Western's tRNA oligos and primer oligos which we approved.
Our oligos were also approved by Missouri Western and Dr. Campbell ordered them while we supervised.

In the afternoon we did the miniprep. The mini prep directions were as appear in the link below.

http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_MiniPrep.html

We then worked with the Nano Prep to dilute our solution of DNA to an absorption of 1.5 and then we made labels for the tubes. The chart below shows relevant information we obtained from the NanoDrop for each of the reporter plasmids.

[[Image: Screen-capture-6.png]]

==Tuesday, June 9, 2009==

'''Preparations for restriction enzymes digestion of plasmids'''

Purpose: To determine the size of the plasmid inserts and ensure that plasmids are what we extracted from the cells during the mini-prep.

Things we need:
DNA ~3uL
Buffer 10x (Want to dilute to 1x) so add 2uL
Enzyme 1 ~1uL
Enzme 2 ~1uL
Water ~13uL
Final Volunter about 20 uL.

Need to choose the right buffer for the restriction enzymes. All buffers come in 10x (10 times more concentrated than what you want it to be when you use it). [http://gcat.davidson.edu/GcatWiki/index.php/Davidson_Missouri_W/Double_Digest_Guide Buffer Website]

Total volume of enzymes has to be 10% or less than final volume (most volume 2uL). Barely submerge the pipet tip into the solution when pipeting the enzymes.
Star (*) activity: Restriction enzyme stops cutting specifically and starts cutting everywhere.

DNA can't be seen by itself so we add EtBr (the bigger the insert, the more will bind) so bigger strands appear brighter. samples.

The order of entry doesn't matter, but make sure to enter the enzymes last.

Will have lots of labeled tubes and will put 3uL of appropriate plasmids into the tubes.
In the meantime, we'll make a "Master Mix" of all the other ingredients in a bigger tube. Always make enough volume for one extra tube.

Enzymes usually have an optimal temperature of 37C. We will let this reaction go for an hour and then we will run it on a gel.

'''Gel Electrophoresis Protocol'''

Website online for ideal gel concentration for resolving different sizes of molecules [http://gcat.davidson.edu/iGEM08/gelwebsite/gelwebsite.html Gel Concentration Website].
See protocol on the Davidson/Missouri Western Wookie for running molecules in the gel and how to digest with restriction enzymes.

'''What's Next?'''

1. 5' end additions
a. Using PCR
b. Cut at the restriction sites EcoR1 and either Bam H1 or Nco1
c. Cut plasmid and reporter with same restriction enzymes as above
d. Run a gel and purify "keeper" DNA (Ligate transform, screen, sequence)
e. Then, we take the "keeper" DNA and combine it with the insert from part

We can have steps d and e ready in advance.

2. tRNAs
a. Assemble the oligos into genes (Requires lots of calculations beforehand, but the actual steps )
b. Plasmids cut up with EcoR1 and Pst1 '''WE CAN USE THE "WASTE" PLASMID FROM THE RESTRICTION ENZYME DIGEST'''
c. Gel purify the plasmids (Ligate transform, screen, sequence)

The following tables include Master Mix measurements, agarose gel concentrations and buffers.
http://gcat.davidson.edu/GcatWiki/images/0/08/Shamitapic3.png
http://gcat.davidson.edu/GcatWiki/images/d/d1/Shamitapic4.png
http://gcat.davidson.edu/GcatWiki/images/6/6f/Shamitapic5.png

We made the Master Mix with our DNA and put it in the incubator at 10:40 AM.

'''Preparing the Gels'''

Now, we will be making 2 gels, 1 at 0.8% and 2.5% (weight to volume percent). The gel volume is 60mL.

'''Definition of 1% solution:
1 gram of agarose/ 100mL buffer'''

So for a 0.8% solution: 0.8 grams agarose/100mL buffer AND we need 60mL (fixed volume for casts run) so multiply that by 0.6.
Final concentration:

.48 grams agarose/60mL buffer

1 uL of EtBr stock for 60mL of gel.
EtBr is in the buffer so it is constantly replenishing itself even though it is moving toward one end in order to make bands on the other end visible.

After Dr. Campbell's demonstration, we each helped prepare a 0.8% buffer solution using the agarose powder, buffer and EtBr. We mixed together the first two substances in a 200mL Erlenmeyer flask and heated it for 120 seconds in the microwave. We then added EtBr and poured the gel solution into the gel cast.

'''Agar Plate'''

After lunch, we grew some more cells for each of the 11 parts in an agar plate labeled "LBAMP-AMC-9 June" so that so would have extra cells that could be stored for up to 2 weeks.
We made small circles on the agar plate and numbered them 1-11. The numbers corresponded to the following parts. We put the plate in the incubator overnight to let the cells grow.

1. B0030 AMC
2. K091111
3. S03710
4. I715039 OEH
5. S03511 ORH
6. I715039 AST
7. B0030 SDP
8. K091112
9. J31007 LJH
10. J31007 REC
11. S03511

'''Unloading the Gels'''

The following shows which parts we entered in which gels:

[[Image: screen-capture-2.png]]

We unloaded the 2.5% agarose first and after observing the bands under a UV light, put the gel into the BioRad machine to observe the bands. The image below is of a Molecular Weight Marker that shows the number of bp in control fragments of DNA according to the distance they travel down the gel.

[[Image: DNA_molecular_weight_marker_gel.png]]

We obtained the following printout for the first gel that was 2.5% Agarose.
[[Image: 2009-06-09_14hr_44min.jpg]]

The bands in the first well at the bottom are those of the molecular weight marker. We skipped the lane above (to the right) of the MWM and entered the rest of the inserts as described in the above table. A light band is visible for each of the 4 other wells. In Lanes 3 and 4 we concluded we are supposed to have approximately 45 bp because the insert is 15bp + 30 bp on either side of the insert (Xba1 and Spe1 restrictions sites + sticky ends + extra bases). We used the same logic for Lanes 5 and 6 which are supposed to contain approximately 86 base pairs.

One discrepancy in this gel, however, is that Lanes 5 and 6, which according to their labels respectively contain the LacIQ promoter and the Lac IQ1 promoter. According to the information online at the parts registry website, pLacIQ1 should be the same length as pLacIQ. The gels do not show this, but instead show that the sample in Lane 5 is smaller than that in Lane 6 because Lane 5 traveled further. Leland Taylor and Romina Clemente sent out an email to Pallavi Penumetcha to ask about this because she had written a paper about this.

We then unloaded the 0.8% agarose gel and then placed this in the BioRad machine under the same protocol as above. The following image is the 0.8% Agarose.

[[Image:2009-06-09_15hr_21min.jpg]]

We observed that the bands in Lane 1 (which is now at the top) are examples of high intensity bands. The plasmids contained in the original well are twice the intensity of the insert.

In Lane 5, however, we observed several different bands. This could be due to the extra DNA that was sitting on the top of the well. We decided to re-expose the gel to get better observations on Lanes 3 and 4 because the bands were not as intense. We anticipated seeing a band at about 250 bp because this was the size of the insert (plus extra bp). After re-exposing the gel, we did not find any difference and still did not see any bands in these lanes. We have decided to redo the gel on the part S03511 and so we prepared the master mix with that DNA and incubated it so that we could run the gel tomorrow.

We also cut out the "empty" plasmids from the gel so that we could use this as vectors for the coding sequence of the suppressor tRNAs.

Tomorrow we will be planning out specific tasks for people once the oligos arrive.

==Wednesday, June 10, 2009==

We began this morning by making the agarose gel with the same protocol as yesterday. This time, since we are just re-doing both S03511 inserts, we will have a 1.8% agarose gel.

'''OUTLINE: WHERE DO WE STAND?'''

Yesterday
a. Isolated empty plasmids
b. Verified most inserts
c. Waiting for:
>tRNA oligos
>Primers
a. Will amplify the reporter gene in the plasmid itself

Next?
a. When Primers Arrive
> Amplify the reporter genes with primers (PCR takes ~3 hours)
> Calculate how much reporter plasmid we will need (Start with 50x more than we actually will need)
> Cut amplified sequence (EcoR1 and BamH1/Nco1) then purify what we need
> Cut reporter plasmid (EcoR1 and BamH1/Nco1) then purify what we need
> Ligate amplified sequence and reporter plasmid
b. When tRNA oligos arrive
> Calculate how much of each oligo we will need (takes a while...)
> "Boil and cool" tRNA oligos together
> Ligate fragments (dsDNA) into plasmids we isolated yesterday (~5 minutes)
> Transform the plasmids (~20 minutes)

* LOOK THROUGH LIGATION PROTOCOL (How to assemble genes)
* LOOK THROUGH TRANSFORMATION PROTOCOL

''What we need to get done today'':
1. Prepare the control for the Tet Resistance by BioBricking the part pLac+RBS (S03511) with the TetA (J31007).
2. Check the part number for RFP and also for pLac+RBS+RFP
3. Read through ligation protocol
4. See how many nanograms of plasmid we will need for the tRNAs and for the inserts and multiply this number by 50.
> Check if we have enough plasmids from our mini-prep
5. Purify the plasmids from the gel

After we made this outline, Leland, Olivia and I analyzed the gel we ran this morning and did not see a more than one band in the 2 lanes that contained DNA from S03511 (image below). Since we did not see multiple bands in yesterday's gel either, we conclude that there is something wrong with the DNA rather than with the restriction enzymes that we used to cut it. We concluded that there is probably not an insert in this plasmid both the part and the plasmid are useless.

[[Image: 2009-06-10 10hr 30min.jpg]]

Now, we do not have an immediately accessible promoter. We have decided to order the pBAD promoter from Missouri Western and we will attach the RBS+Tet to this plasmid. Our insert for this will be the larger gene because it is easier to manipulate in the gel than the small gene.

Alyndria Thompson and Romina Clemente are figuring out the size of the plasmids including the reporter genes.

After lunch we performed several steps to isolate and purify the gel to get the empty plasmids will be the vectors for the tRNA DNA. Then, we used the NanoDrop to assess the purity of our DNA. We found that the plasmids were more pure than we had expected. The following values were obtained:

Abs 1.850
A-260 10 mm path 0.172
A-280 10 mm path 0.075
260/280 2.28
260/230 0.09

We then followed the protocol for preparing the agar plates. We put the plate with the agar solution in the Autoclave.

While the solution was in the autoclave, we discussed that we don't really need a promoter in front of RBS+Tet for the gene to be expressed because Tet expression is so strong that the gene would translate without a promoter. However, it would be a good idea to have the promoter because if it was possible to reinforce the translation of the reporter protein, we wanted to do that.

We found that there were some tubes in the lab fridge that contained the pBAD and pLac promoters with RBS. We decided to use these as the promoter and RBS for our experiments. The pLac promoter will be used for RFP and the pBad promoter used with Tet Resistance. We decided to use the pBad with Tet because it is weaker in comparison to pLac. As expression of the Tet gene can be toxic to a cell, we do not want to overexpress the gene with a stronger promoter.

Once the agar solution was sterilized in the Autoclave, we removed it, cooled it, and pipetted the antibiotic resistance into it. We then poured the solution into the plates, removed any bubbles, and let the gel form.

Alyndria Thompson and Romina Clemente concluded that we had more than enough recieving vectors for both the RFP and Tetracycline genes. Since we need 50 nanograms, it is wise to have at least 50 times that much that we can work with, so we need at least 2,500 ng.

RFP 1: 77.6 x 4 = 310.4 ng/uL
x 20 uL = 3,272 ng
RFP 2: 59.5 x 4 = 238 ng/uL
x 20 uL = 4,760 ng
Tet: 40.9 x 4 = 163.6 ng/uL
x 20 uL = 3,272 ng

According to these calculations we have more than 50 times what we need for both instances of RFP and Tet.

Tomorrow, we can prepare the Master Mix for the digestions of both reporter plamids and then add the restriction enzymes once they arrive. Then, we can follow through with the rest of the plans we had made above in the section titles "Next?".

==Thursday, June 11, 2009==

We began this morning by calculating how many uL we would need for each of the mixes for digesting our reporter plasmids. We decided to double the final volume of each mix to 40uL instead of 20uL. Since we knew the concentration of each plasmid and also knew we needed 2500 ng, we cross multiplied to find the uL of DNA we would need for each.

Concentration Calculations:
310.4 ng/1 uL = 2500ng/x uL
x = 8.05 uL

Here is a chart indicating how much of each we used for the 2 mixes.

[[Image: DoubledigestRFPTetR.png]]

On the Promega website, we found a list of compatible buffers for double digestions. Buffers E and H are compatible for the Tet plasmid and RFP plasmid digestions respectively. We fortunately have both buffers in stock.

[[Image:BamEcoR1.png]] [[Image:NcoEcoR1.png]]

We waited for our waited for the restriction enzymes to arrive and in the meantime, we transformed the plasmids with pLac+RBS, pBad, and pBad+RBS into ''E.coli'' cells. We want the ''E.coli'' cells to replicate the plasmids with these genes because we have a limited amount of genes. [http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_Transformation.html Zippy Plasmid Transformation]

Once our oligos and restriction enzymes arrived, we got to work right away!

'''PCR (Polymerase Chain Reaction)'''

We calculated how much plasmid we will need for the PCR. We decided to dilute the concentration of our plasmids a hundred fold so that we could get pipetable amounts.

''Change in Concentrations''
310.4 ng/uL x 1/100 (dilution factor) = 3.104 ng/uL (RFP)
163.6 ng/uL x 1/100 (dilution factor) = 0.164 ng/uL (Tet)

''Example shown with RFP''
3.104 ng/1 uL = 1ng/ x uL, x = .32
For Tet, x = .61

We also calculated how much of each primer we would need with the following equation:

C1V1 = C2V2
(100 uM) (x) = (1 uM)( 100 uL)
x = 1 uL

[[Image: screen-capture-8.png]]

Each of the 5 of us made had 2 tubes (one for each reporter protein) because there were 5 different forward primers.To each tube, we added the template DNA (either RFP or Tet), water, the respective reverse primer (either RFP or Tet), and the forward primer depending on which 5mer we had chosen. My 5mer was CCAUC. Finally, we added the Master Mix last because this contained the enzyme (DNA polymerase) that would amplify our desired sequence.

[[Image: Pcrtubes.png]]

We followed the temperature cycles so that PCR could occur. The annealing temperature needed to be 5C less than the smallest melting temperature of the primers. Since the reverse primer of RFP had a melting temp of 66C, we set the temperature of the 3rd cycle on 61C.

[[Image: Temperaturecycle.png]]

'''tRNA Oligo Assembly'''

We needed to calculate the concentration of each of the oligo before carrying out the assembly. The stock concentration of each of the oligo was 100 uM. Since we wanted to have 5uM of the oligo in the test tube, and we know that final volume of the test tube is 20 uL, we just cross multiplied to find the amount we needed to add to the tubes to get this concentration.

(100uM)x = (5uM)(20uL)
x = 1 uL

We added 1 uL of each oligo to the tubes.

We had six total tRNAs we needed to assemble so each of us took one FML (one person took two) and to each tube we added 1uL of each oligo for a total of 7uL, 2uL of buffer, and 11uL of water for a total volume of 20 uL. Five of the oligos were constant and 2 varied for each of the different tRNAs. So everyone pipetted the same 5 oligos and everyone also had 2 unique oligos to add. When all of the oligos were added and the tubes ready, we boiled the tubes at 50C for 5 minutes. We then allowed the tubes to remain in the water to cool very slowly overnight so that the annealing process could take place.

The last thing we did at the end of the day was finish off our plasmid digestion that we had begun in the morning. We added the buffer, DNA, water, and restriction enzymes and placed this in the incubator for overnight digestion.

'''To Do''':

1. Isolate our desired dsDNA from the PCR.
2. Verify our tRNA inserts and eventually transform these into plasmids.
3. Check our agar plates to see colonies that have replicated our pLac+RBS, pBAD+RBS, and pBAD genes.

==Friday, June 12, 2009==

This morning, we began by outlining the main items on today's agenda.

1. tRNAs: Ligation, Transformation
2. Digestion reporters (run all of it and gel purify)
3. Transformed cells
4. PCR: Gel run an aliquot to verify (5uL)

We used the equation given by the Ligation protocol to calculate how many nanograms of insert we would need. Initially, we started out calculated the MW average of the 9 and 10 anticodon loops of tRNAs which were 15828.23 ug/umols and 159016.6 ug/umols respectively. We then saw, however, that these would not be helpful values in the process. Instead we started out with the equation below to find the final ng of insert we would need for the ligation.

[[Image: ligationcalculations.png]]

We calculated our initial concentration in ng/uL of insert as shown below.

[[Image: ligationequation.png]]
X nanograms of insert = [(2)(144bp)(50 ng linearalized plasmid)]/2038bp
X = 7.0657 ng of insert

We wanted to find the amount of uL of insert we would need to use. We diluted the initial concentration 50x because we wanted a pipettable amount of insert. Since we knew we wanted the final amount of nanograms to be 7.0657, we calculated as shown below:

''DILUTION'': 555.48/50 = 11.012
''CALCULATION'': 11.012 ng/ 1uL = 7.065 ng/ x uL
x = .64 uL of insert

After we had done these calculations, half of the group started to carryout the ligation of the tRNA inserts into empty plasmids. We needed 7 ligations total: 6 were the tRNAs we needed, while the last one is a negative control. These are the final amounts of what we used:

[[Image: Trnaligation.png]]

The other half of the group (which included me) prepared the agarose gels so that we could run gels for the digested reporters and the PCR inserts. We calculated that the digested reporters needed an agarose of 4% and the PCR reporters needed an agarose of 1.45%. We allowed this gel to form for about 45 minutes to an hour. We prepared both the gels and allowed them to run through lunch.
After lunch, we took pictures of our gels and noticed that the third lane in the PCR gel (third lane from the top in the bottom photo) did not develop a band. The lane was supposed to contain the CUAGU insert for Tetracycline Resistance.

[[Image: 2009-06-12 15hr 54min.jpg]] [[Image:2009-06-12_16hr_07min.jpg]]

Romina Clemente and I decided to repeat this PCR using the TetResistance and RBS+TetResistance plasmids as templates. We labeled J31007 as "2*" and S03710 as "2!". We had to recalculate how much template DNA to use. We let the PCR run overnight. We also verified that the digestion fragments (both the plasmid and the insert) on the other gel were the correct size. The plasmids, which are the brighter fragment in the gel with only 2 lanes, contain the plasmid base pairs along with the rest of our reporter genes. We isolated these plasmids from the gel today and stored them for purification on Monday.

In the meantime, other members of our lab had finished the ligation of the tRNA genes to the empty plasmids and were now transforming these into competent cells. They placed the agar plate with these cells in the fridge overnight.

We also observed the transformations that we had performed yesterday and noticed that I13453 did not form any colonies. J04500 and J3102 did form colonies.

To Do:
1. PCR: Dig, Ligate, Transform
Clean and concentration column, digest, clean and concentration column, ligate.
2. Gel purification of our digestion plasmids
3. Check if colonies have grown for our tRNAs

==Saturday, June 13, 2009==

Alyndria Thompson came in on Saturday morning to remove the tRNA plates and put the PCR reactions in the fridge.

'''tRNA Colonies'''

She counted the colonies of tRNA on each plate.

[[Image:TRNA_colonies_6_13_09.png]]

She placed the plates in the large fridge on the top shelf at 9:23 am. She talked to Dr. Campbell who said we will have to try to "work them up".

'''PCR'''

Products were placed in orange rack of smaller fridge. [2*, 2!]

==Sunday, June 14, 2009==

We decided to re-ligate and re-transform all of the tRNA inserts because we did not get very good yields of colonies from the transformations.
We are using the same measurements for this ligation as we had used on Friday.

[[Image: Trnaligation.png]]

We also stored liquid cultures of the colonies that greater than a 2:1 variable:negative control ratio just in case we wanted to use them for mini preps in the future.

IGEM Notebook

2009-06-15T01:57:51Z

ShPunjabi: /* Saturday, June 13, 2009 */

==Wednesday, June 3, 2009==

Romina Clemente and I are trying to find suitable reporter proteins to use. Yesterday, Leland Taylor and Alyndria Thompson were working on ways to insert the gene sequences into the plasmid. Upon seeing how they wanted to manipulated the reporter gene to include the logical clauses, we came up with a few criteria for the reporter genes we would use. The following criteria for genes are listed in order of the broadest aspect to look at to the narrowest aspect:
a) Doesn't contain restriction sites for the 4 restriction enzymes (EcoR1, Xbal, Spel, Pst1) used to cleave the Biobrick part out of the plasmid.
b) Contains 6 cutter restriction sites.
c) These restriction enzymes aren't blunt (cleave straight down at one spot).
d) These restriction sites are close to thge 5' (beginning) end of the sequence.
e) These enzymes are easiest to work with and cheapest.
We are finding the part numbers of the reporter genes we want to use (antibiotic resistance, fluorescence, LacZ) through our own GCAT because we know these ones work. We are then locating these parts on the parts registry [1] website. We copied and pasted the gene sequences we obtained from the registry onto the ApE software [2]. From here, we were able to generate a genetic map of each gene that outlined each restriction site that fit our criteria. We put each genetic map, alongside the part number used, into a Word document.

http://gcat.davidson.edu/GcatWiki/images/0/0e/Restriction_Site_Mapping_on_Reporter_Genes.doc

Later on in the day, we decided that the best way to test for suppression would involve placing the 5mers at the beginning of the reporter gene instead of inserting them into the reporter gene. To do this, we decided that the gene along with its start codon need to be expressed after the 5mer. We will insert a BioBrick plasmid with a 5mer and start codon that will be incorporated before the gene:
RBS-6-8nt-ATG-5mer--ATG-gene

Leland Taylor assembled the oligos that we will need for the BioBrick pieces.

In order to test whether the reporter protein would be expressed regardless of the 5mer being suppressed or not, we would need to remove the suppressor tRNAs from the cell.

Meanwhile, I helped figure out the coding sequence that we will need for the suppressor tRNAs that will bind to the 5mers. We decided that we would be using the following suppressor tRNAs on the Davidson side: CUAGU, CCCUC, CGGUC, CCAUC, and CCACU. We searched through several papers, with little result and finally emailed Dr. Christopher Anderson of UC Berkeley to request the DNA sequence. He emailed us back with a generic DNA code that we could use to create the tRNA; however, for each suppressor tRNA we would use, we would need to change base pairs in the anticodon loop.

The length of the tRNA is 92 nt. The anticodon loop is comprised of 9 nt. Once we have BioBricked the DNA sequence with 4 restriction sites and supplemental nucleotides, the length of the entire gene is 144 nucleotides. This is made up of the 92 nt +22 nt on each side of the sequence (44) + 4 nt on each side of the restriction ends (8).

At the end of the day, we decided to use tetracycline resistance and RFP as our reporter proteins for this tester experiment.

==Thursday, June 4, 2009==

This morning, I began the day by learning how to use the PEARL prgram. Olivia Ho-Shing sat with me and walked me through the program script. And then, Leland Taylor and Shashank Suresh had a problem they wanted us to solve. Leland explained that we need to make sure there is a stop codon in the gene if we fail to suppress the 5mer. The stop codon needs to be before we find another 5mer. They also wanted us to run through both the RFP and Tet Resistance genes to make sure that they did not contain any of the anticodon sequences from our 5 frameshift suppressor tRNAs so as to throw the translation out of frame. They further wanted us to confirm that if we do have successful suppression, there are no stop codons in the middle of the RFP and Tet Resistance genes. PEARL did not find any matches for the suppressor tRNAs within genes. We found 3 matches for stop codons UAA. Two of these occured at the end of the Tet and one at the end of RFP, where they should normally be to end translation into a protein.

Following this, Olivia Ho-Shing and I tried to determine how we can add BioBrick ends to our single stranded DNA sequences that code for the suppressor tRNAs. Our ultimate goal is to find complements for these single strands so they can be put into plasmids. We found the standard prefixes and suffixes for sequences that do not contain ATG (becuase this sequence is a functional RNA and will not be translated) and we placed these before and after the altered 92bp sequence that Dr. Anderson provided us with. The prefixes and suffixes are necessary because they are the extra nucleotides that will be mimicking a "restriction enzyme cutting site". The single strand appeared as below after the Bio Brick prefixes and suffixes were added:

22ntPREFIX--32nt--VARIABLE ANTICODON LOOP--51nt--21ntSUFFIX

We used the suppressor codon CUAGU as our example and placed its appropriate anticodon loop in the sequence. We put this into the lancelator that would give us the other strand as well as the oligos we would need for ideal construction of this double stranded DNA fragment to put into the plasmid. We recieved a total of 4 oligos, 2 of which were variable and 2 of which were constant for all 6 different suppressor tRNAs. Using this double strand, we simulated a restriction enzyme digestion by EcoRI and PstI. We took off the G at both ends of the top strand and then removed the first five nucleotides on each end of the bottom strand. This made the top strand longer than the bottom one. We then proceeded to take the two variable strands and changed the anticodon loop nt for each suppressor tRNA we used.

Around lunchtime, we encountered a problem with the BioBrick scar in the "ATG-5mer subpiece" of the test project. It turns out that after annealing the sticky ends of the Xba1 and Spe1 sites, the scar created had a stop codon TAG (UAG) that was in frame with the suppressed codon. When the Xba1 site on insert anneals to the Spe1 site on the plasmid (the green portions), we get a scar that reads TACTAG.
[[Image:screen-capture.png]]

For the afternoon, Olivia Ho-Shing and I finished assembly of the oligos of the suppressor tRNA codes. We assembled the document below:

http://gcat.davidson.edu/GcatWiki/images/5/5e/TRNAoligostoOrder.doc

We were assigned tasks of creating "cartoons" to illustrate the phenomenon that had occurred. No one could successfully do this however, because we could not understand how the piece had formed. The parts registry website provided wrong information on the restriction site of Spe1 which slowed our efficiency. This project turned into an effort to try to find two other different restriction enzymes that have complementary sticky ends other than Xba1 and Spe1. We want to get rid oc the scar that contains TACTAG.

We ended up deciding that a hybrid of two ideas was needed: restriction enzymes would need to be used to make several reporter gene sequences with different beginnings dependending on the suppressor tRNA sequence needed.

==Friday, June 5, 2009==

Our goal for this morning was to discuss the hybrid PCR approach to assemble the part that comes before the gene we wanted to express. We decided that we would have a BioBrick prefix and a 5mer followed by a sequence of nucleotides whose identity was up to us. Using PCR we would get the second double strand if we have the following:

BioBrick Prefix--ATG--5mer--gene until we get to restriction site

The PCR primer would attach to the ATG and the 5mer and would replicate the complementary base pairs to make a double stranded DNA sequence. As several of us were confused about PCR methods, we looked at the online link provided by Alyndria Thompson's link to the PCR interactive website which was very helpful. We also did some research to discover that the restriction enzyme BseRI (which is contained in the YFP that we wanted to alter) is not compatible with any other restriction enzyme. This is good because we did not want to plasmid containing this gene to close up with Xba1 once we digested it.

The team experienced some confusion with the hybrid idea and spoke to Clif Davis of Missouri Western University on the phone. He told us that there will be one part that is a fragment of the beginning of some nucleotides as a restriction cleaving site, ATG, the logical clause, and the start of the YFP gene that will be placed into a plasmid containing a space. To the left of the space will be the BioBrick Prefix and the right of the space will contain the rest of the genes in the YFP. Therefore, we decided not to alter any BioBrick ends and just look at restriction sites at the beginning of reporter proteins. The small fragment that will be placed into the plasmid will be made by PCR. The PCR will attach a primer onto the ATG and 5mer and replicate.

While we were discussing this, we expanded upon the idea that we can do whatever we want with the "suffix" portion between the 5mer and the beginning of YFP. We decided it would be best to remove ATG and add the first 5 amino acids and the restriction enzyme site. Along with this, if we have the 5mer coding for a certain amino acid (ie methionine) we could replace another codon that codes for methionine WITH that 5mer so long as this codon is contained within the first 15 nt. This way, we would not be altering the protein.

Olivia Ho-Shing came up with an idea of cascading PCR effect for longer logical clauses. Post translational modification of the protein was another one of her ideas. This applies because although we may code for the protein in a given way, the protein may modify or fix itself up to perform a slightly different function in its tertiary form.

Dr. Campbell suggested that we can look up restriction enzymes 400-500 base pairs into the reporter protein we would like to use; however, we have more flexibility in our procedure if we choose more "hardy" cutters. So instead, we could cut with EcoRI instead of with Xba1.

During the afternoon, we set about three tasks:
1) Which primers to use
2) Which promoters to use
3) Which restriction enzymes and how they will determine use of reporter proteins

Olivia Ho-Shing and I began looking up which restriction enzymes were strong so we could base our reporters on that. We began with Tet and RFP since those were the two reporters we had started out with choosing. I found a website that provided enzyme cleavage efficiency based upon the proximity of the site to the end of the PCR fragment that contains it. We discovered the following based upon the attached documents:

http://gcat.davidson.edu/GcatWiki/images/8/8f/Restriction_Enzyme_Cleavage_Efficiency.doc
http://gcat.davidson.edu/GcatWiki/images/9/9a/07Jun09_RE_Cutting_Efficiency_oh.doc

We decided that these two reporters contained efficient enzymes in a good quantity to choose from. On Monday, we will look through these enzymes once more and finalize which enzymes we would like to use with RFP and Tet.

==Sunday, June 7, 2009==

We did preparations for the mini prep this evening. The following notes are courtesy of Olivia Ho-Shing.

1. Sterically transfer ~2mL LB+AMP (AMP are ampicillin resistant plasmids) *Never put fluid back into bottle once it has entered pipette*
2. Use forceps to grab toothpick, scrap some frozen cells, rub in tube with top

'''Tubes'''

S03511 (2)

K091111

B0030

I715039

K091112

I715039

J31007 (2)

B0034

E1010

S03710

==Monday, June 8, 2009==

We reviewed the PCR process this morning as well as the restriction enzyme cleavage process.

Then, Leland Taylor, Olivia Ho-Shing and I all worked on converting the tRNA oligos we made to the template that Missouri Western used. The oligos we had previously made were different from the ones that Dr. Anderson had originally emailed us because further communication between Dr. Anderson and Missouri Western resulted in a better template.

We tried several times to minimize the number of oligos while keeping their individual lengths under 80bp. We managed to do this in the end by creating 7 oligos, 2 of which are variable between the suppressor tRNAs.
We reviewed the following document and finalized the oligos that we needed to order for the frameshift suppressor tRNAs. We decided to keep both the 10bp and 9bp anticodons of CCAUC.

I also looked up the amino acids that corresponded to the FS (frameshift suppressor) tRNAs for future use:

1. CUAGU = Leu
2. CCCUC = Pro
3. CGGUC = Arg
4. CCACU = Pro
5. CCAUC* = Pro
*9 and 10 bp anticodons

Also, we have decided to use NcoI as the restriction enzyme for RFP. This cleavage site occurs 419 bp into the gene. We have decided to use BamHI as the enzyme for Tetracycline Resistance and the cleavage site occurs 290bp into the gene. Both of these enzymes are very powerful and hardy.

We also looked over Missouri Western's tRNA oligos and primer oligos which we approved.
Our oligos were also approved by Missouri Western and Dr. Campbell ordered them while we supervised.

In the afternoon we did the miniprep. The mini prep directions were as appear in the link below.

http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_MiniPrep.html

We then worked with the Nano Prep to dilute our solution of DNA to an absorption of 1.5 and then we made labels for the tubes. The chart below shows relevant information we obtained from the NanoDrop for each of the reporter plasmids.

[[Image: Screen-capture-6.png]]

==Tuesday, June 9, 2009==

'''Preparations for restriction enzymes digestion of plasmids'''

Purpose: To determine the size of the plasmid inserts and ensure that plasmids are what we extracted from the cells during the mini-prep.

Things we need:
DNA ~3uL
Buffer 10x (Want to dilute to 1x) so add 2uL
Enzyme 1 ~1uL
Enzme 2 ~1uL
Water ~13uL
Final Volunter about 20 uL.

Need to choose the right buffer for the restriction enzymes. All buffers come in 10x (10 times more concentrated than what you want it to be when you use it). [http://gcat.davidson.edu/GcatWiki/index.php/Davidson_Missouri_W/Double_Digest_Guide Buffer Website]

Total volume of enzymes has to be 10% or less than final volume (most volume 2uL). Barely submerge the pipet tip into the solution when pipeting the enzymes.
Star (*) activity: Restriction enzyme stops cutting specifically and starts cutting everywhere.

DNA can't be seen by itself so we add EtBr (the bigger the insert, the more will bind) so bigger strands appear brighter. samples.

The order of entry doesn't matter, but make sure to enter the enzymes last.

Will have lots of labeled tubes and will put 3uL of appropriate plasmids into the tubes.
In the meantime, we'll make a "Master Mix" of all the other ingredients in a bigger tube. Always make enough volume for one extra tube.

Enzymes usually have an optimal temperature of 37C. We will let this reaction go for an hour and then we will run it on a gel.

'''Gel Electrophoresis Protocol'''

Website online for ideal gel concentration for resolving different sizes of molecules [http://gcat.davidson.edu/iGEM08/gelwebsite/gelwebsite.html Gel Concentration Website].
See protocol on the Davidson/Missouri Western Wookie for running molecules in the gel and how to digest with restriction enzymes.

'''What's Next?'''

1. 5' end additions
a. Using PCR
b. Cut at the restriction sites EcoR1 and either Bam H1 or Nco1
c. Cut plasmid and reporter with same restriction enzymes as above
d. Run a gel and purify "keeper" DNA (Ligate transform, screen, sequence)
e. Then, we take the "keeper" DNA and combine it with the insert from part

We can have steps d and e ready in advance.

2. tRNAs
a. Assemble the oligos into genes (Requires lots of calculations beforehand, but the actual steps )
b. Plasmids cut up with EcoR1 and Pst1 '''WE CAN USE THE "WASTE" PLASMID FROM THE RESTRICTION ENZYME DIGEST'''
c. Gel purify the plasmids (Ligate transform, screen, sequence)

The following tables include Master Mix measurements, agarose gel concentrations and buffers.
http://gcat.davidson.edu/GcatWiki/images/0/08/Shamitapic3.png
http://gcat.davidson.edu/GcatWiki/images/d/d1/Shamitapic4.png
http://gcat.davidson.edu/GcatWiki/images/6/6f/Shamitapic5.png

We made the Master Mix with our DNA and put it in the incubator at 10:40 AM.

'''Preparing the Gels'''

Now, we will be making 2 gels, 1 at 0.8% and 2.5% (weight to volume percent). The gel volume is 60mL.

'''Definition of 1% solution:
1 gram of agarose/ 100mL buffer'''

So for a 0.8% solution: 0.8 grams agarose/100mL buffer AND we need 60mL (fixed volume for casts run) so multiply that by 0.6.
Final concentration:

.48 grams agarose/60mL buffer

1 uL of EtBr stock for 60mL of gel.
EtBr is in the buffer so it is constantly replenishing itself even though it is moving toward one end in order to make bands on the other end visible.

After Dr. Campbell's demonstration, we each helped prepare a 0.8% buffer solution using the agarose powder, buffer and EtBr. We mixed together the first two substances in a 200mL Erlenmeyer flask and heated it for 120 seconds in the microwave. We then added EtBr and poured the gel solution into the gel cast.

'''Agar Plate'''

After lunch, we grew some more cells for each of the 11 parts in an agar plate labeled "LBAMP-AMC-9 June" so that so would have extra cells that could be stored for up to 2 weeks.
We made small circles on the agar plate and numbered them 1-11. The numbers corresponded to the following parts. We put the plate in the incubator overnight to let the cells grow.

1. B0030 AMC
2. K091111
3. S03710
4. I715039 OEH
5. S03511 ORH
6. I715039 AST
7. B0030 SDP
8. K091112
9. J31007 LJH
10. J31007 REC
11. S03511

'''Unloading the Gels'''

The following shows which parts we entered in which gels:

[[Image: screen-capture-2.png]]

We unloaded the 2.5% agarose first and after observing the bands under a UV light, put the gel into the BioRad machine to observe the bands. The image below is of a Molecular Weight Marker that shows the number of bp in control fragments of DNA according to the distance they travel down the gel.

[[Image: DNA_molecular_weight_marker_gel.png]]

We obtained the following printout for the first gel that was 2.5% Agarose.
[[Image: 2009-06-09_14hr_44min.jpg]]

The bands in the first well at the bottom are those of the molecular weight marker. We skipped the lane above (to the right) of the MWM and entered the rest of the inserts as described in the above table. A light band is visible for each of the 4 other wells. In Lanes 3 and 4 we concluded we are supposed to have approximately 45 bp because the insert is 15bp + 30 bp on either side of the insert (Xba1 and Spe1 restrictions sites + sticky ends + extra bases). We used the same logic for Lanes 5 and 6 which are supposed to contain approximately 86 base pairs.

One discrepancy in this gel, however, is that Lanes 5 and 6, which according to their labels respectively contain the LacIQ promoter and the Lac IQ1 promoter. According to the information online at the parts registry website, pLacIQ1 should be the same length as pLacIQ. The gels do not show this, but instead show that the sample in Lane 5 is smaller than that in Lane 6 because Lane 5 traveled further. Leland Taylor and Romina Clemente sent out an email to Pallavi Penumetcha to ask about this because she had written a paper about this.

We then unloaded the 0.8% agarose gel and then placed this in the BioRad machine under the same protocol as above. The following image is the 0.8% Agarose.

[[Image:2009-06-09_15hr_21min.jpg]]

We observed that the bands in Lane 1 (which is now at the top) are examples of high intensity bands. The plasmids contained in the original well are twice the intensity of the insert.

In Lane 5, however, we observed several different bands. This could be due to the extra DNA that was sitting on the top of the well. We decided to re-expose the gel to get better observations on Lanes 3 and 4 because the bands were not as intense. We anticipated seeing a band at about 250 bp because this was the size of the insert (plus extra bp). After re-exposing the gel, we did not find any difference and still did not see any bands in these lanes. We have decided to redo the gel on the part S03511 and so we prepared the master mix with that DNA and incubated it so that we could run the gel tomorrow.

We also cut out the "empty" plasmids from the gel so that we could use this as vectors for the coding sequence of the suppressor tRNAs.

Tomorrow we will be planning out specific tasks for people once the oligos arrive.

==Wednesday, June 10, 2009==

We began this morning by making the agarose gel with the same protocol as yesterday. This time, since we are just re-doing both S03511 inserts, we will have a 1.8% agarose gel.

'''OUTLINE: WHERE DO WE STAND?'''

Yesterday
a. Isolated empty plasmids
b. Verified most inserts
c. Waiting for:
>tRNA oligos
>Primers
a. Will amplify the reporter gene in the plasmid itself

Next?
a. When Primers Arrive
> Amplify the reporter genes with primers (PCR takes ~3 hours)
> Calculate how much reporter plasmid we will need (Start with 50x more than we actually will need)
> Cut amplified sequence (EcoR1 and BamH1/Nco1) then purify what we need
> Cut reporter plasmid (EcoR1 and BamH1/Nco1) then purify what we need
> Ligate amplified sequence and reporter plasmid
b. When tRNA oligos arrive
> Calculate how much of each oligo we will need (takes a while...)
> "Boil and cool" tRNA oligos together
> Ligate fragments (dsDNA) into plasmids we isolated yesterday (~5 minutes)
> Transform the plasmids (~20 minutes)

* LOOK THROUGH LIGATION PROTOCOL (How to assemble genes)
* LOOK THROUGH TRANSFORMATION PROTOCOL

''What we need to get done today'':
1. Prepare the control for the Tet Resistance by BioBricking the part pLac+RBS (S03511) with the TetA (J31007).
2. Check the part number for RFP and also for pLac+RBS+RFP
3. Read through ligation protocol
4. See how many nanograms of plasmid we will need for the tRNAs and for the inserts and multiply this number by 50.
> Check if we have enough plasmids from our mini-prep
5. Purify the plasmids from the gel

After we made this outline, Leland, Olivia and I analyzed the gel we ran this morning and did not see a more than one band in the 2 lanes that contained DNA from S03511 (image below). Since we did not see multiple bands in yesterday's gel either, we conclude that there is something wrong with the DNA rather than with the restriction enzymes that we used to cut it. We concluded that there is probably not an insert in this plasmid both the part and the plasmid are useless.

[[Image: 2009-06-10 10hr 30min.jpg]]

Now, we do not have an immediately accessible promoter. We have decided to order the pBAD promoter from Missouri Western and we will attach the RBS+Tet to this plasmid. Our insert for this will be the larger gene because it is easier to manipulate in the gel than the small gene.

Alyndria Thompson and Romina Clemente are figuring out the size of the plasmids including the reporter genes.

After lunch we performed several steps to isolate and purify the gel to get the empty plasmids will be the vectors for the tRNA DNA. Then, we used the NanoDrop to assess the purity of our DNA. We found that the plasmids were more pure than we had expected. The following values were obtained:

Abs 1.850
A-260 10 mm path 0.172
A-280 10 mm path 0.075
260/280 2.28
260/230 0.09

We then followed the protocol for preparing the agar plates. We put the plate with the agar solution in the Autoclave.

While the solution was in the autoclave, we discussed that we don't really need a promoter in front of RBS+Tet for the gene to be expressed because Tet expression is so strong that the gene would translate without a promoter. However, it would be a good idea to have the promoter because if it was possible to reinforce the translation of the reporter protein, we wanted to do that.

We found that there were some tubes in the lab fridge that contained the pBAD and pLac promoters with RBS. We decided to use these as the promoter and RBS for our experiments. The pLac promoter will be used for RFP and the pBad promoter used with Tet Resistance. We decided to use the pBad with Tet because it is weaker in comparison to pLac. As expression of the Tet gene can be toxic to a cell, we do not want to overexpress the gene with a stronger promoter.

Once the agar solution was sterilized in the Autoclave, we removed it, cooled it, and pipetted the antibiotic resistance into it. We then poured the solution into the plates, removed any bubbles, and let the gel form.

Alyndria Thompson and Romina Clemente concluded that we had more than enough recieving vectors for both the RFP and Tetracycline genes. Since we need 50 nanograms, it is wise to have at least 50 times that much that we can work with, so we need at least 2,500 ng.

RFP 1: 77.6 x 4 = 310.4 ng/uL
x 20 uL = 3,272 ng
RFP 2: 59.5 x 4 = 238 ng/uL
x 20 uL = 4,760 ng
Tet: 40.9 x 4 = 163.6 ng/uL
x 20 uL = 3,272 ng

According to these calculations we have more than 50 times what we need for both instances of RFP and Tet.

Tomorrow, we can prepare the Master Mix for the digestions of both reporter plamids and then add the restriction enzymes once they arrive. Then, we can follow through with the rest of the plans we had made above in the section titles "Next?".

==Thursday, June 11, 2009==

We began this morning by calculating how many uL we would need for each of the mixes for digesting our reporter plasmids. We decided to double the final volume of each mix to 40uL instead of 20uL. Since we knew the concentration of each plasmid and also knew we needed 2500 ng, we cross multiplied to find the uL of DNA we would need for each.

Concentration Calculations:
310.4 ng/1 uL = 2500ng/x uL
x = 8.05 uL

Here is a chart indicating how much of each we used for the 2 mixes.

[[Image: DoubledigestRFPTetR.png]]

On the Promega website, we found a list of compatible buffers for double digestions. Buffers E and H are compatible for the Tet plasmid and RFP plasmid digestions respectively. We fortunately have both buffers in stock.

[[Image:BamEcoR1.png]] [[Image:NcoEcoR1.png]]

We waited for our waited for the restriction enzymes to arrive and in the meantime, we transformed the plasmids with pLac+RBS, pBad, and pBad+RBS into ''E.coli'' cells. We want the ''E.coli'' cells to replicate the plasmids with these genes because we have a limited amount of genes. [http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_Transformation.html Zippy Plasmid Transformation]

Once our oligos and restriction enzymes arrived, we got to work right away!

'''PCR (Polymerase Chain Reaction)'''

We calculated how much plasmid we will need for the PCR. We decided to dilute the concentration of our plasmids a hundred fold so that we could get pipetable amounts.

''Change in Concentrations''
310.4 ng/uL x 1/100 (dilution factor) = 3.104 ng/uL (RFP)
163.6 ng/uL x 1/100 (dilution factor) = 0.164 ng/uL (Tet)

''Example shown with RFP''
3.104 ng/1 uL = 1ng/ x uL, x = .32
For Tet, x = .61

We also calculated how much of each primer we would need with the following equation:

C1V1 = C2V2
(100 uM) (x) = (1 uM)( 100 uL)
x = 1 uL

[[Image: screen-capture-8.png]]

Each of the 5 of us made had 2 tubes (one for each reporter protein) because there were 5 different forward primers.To each tube, we added the template DNA (either RFP or Tet), water, the respective reverse primer (either RFP or Tet), and the forward primer depending on which 5mer we had chosen. My 5mer was CCAUC. Finally, we added the Master Mix last because this contained the enzyme (DNA polymerase) that would amplify our desired sequence.

[[Image: Pcrtubes.png]]

We followed the temperature cycles so that PCR could occur. The annealing temperature needed to be 5C less than the smallest melting temperature of the primers. Since the reverse primer of RFP had a melting temp of 66C, we set the temperature of the 3rd cycle on 61C.

[[Image: Temperaturecycle.png]]

'''tRNA Oligo Assembly'''

We needed to calculate the concentration of each of the oligo before carrying out the assembly. The stock concentration of each of the oligo was 100 uM. Since we wanted to have 5uM of the oligo in the test tube, and we know that final volume of the test tube is 20 uL, we just cross multiplied to find the amount we needed to add to the tubes to get this concentration.

(100uM)x = (5uM)(20uL)
x = 1 uL

We added 1 uL of each oligo to the tubes.

We had six total tRNAs we needed to assemble so each of us took one FML (one person took two) and to each tube we added 1uL of each oligo for a total of 7uL, 2uL of buffer, and 11uL of water for a total volume of 20 uL. Five of the oligos were constant and 2 varied for each of the different tRNAs. So everyone pipetted the same 5 oligos and everyone also had 2 unique oligos to add. When all of the oligos were added and the tubes ready, we boiled the tubes at 50C for 5 minutes. We then allowed the tubes to remain in the water to cool very slowly overnight so that the annealing process could take place.

The last thing we did at the end of the day was finish off our plasmid digestion that we had begun in the morning. We added the buffer, DNA, water, and restriction enzymes and placed this in the incubator for overnight digestion.

'''To Do''':

1. Isolate our desired dsDNA from the PCR.
2. Verify our tRNA inserts and eventually transform these into plasmids.
3. Check our agar plates to see colonies that have replicated our pLac+RBS, pBAD+RBS, and pBAD genes.

==Friday, June 12, 2009==

This morning, we began by outlining the main items on today's agenda.

1. tRNAs: Ligation, Transformation
2. Digestion reporters (run all of it and gel purify)
3. Transformed cells
4. PCR: Gel run an aliquot to verify (5uL)

We used the equation given by the Ligation protocol to calculate how many nanograms of insert we would need. Initially, we started out calculated the MW average of the 9 and 10 anticodon loops of tRNAs which were 15828.23 ug/umols and 159016.6 ug/umols respectively. We then saw, however, that these would not be helpful values in the process. Instead we started out with the equation below to find the final ng of insert we would need for the ligation.

[[Image: ligationcalculations.png]]

We calculated our initial concentration in ng/uL of insert as shown below.

[[Image: ligationequation.png]]
X nanograms of insert = [(2)(144bp)(50 ng linearalized plasmid)]/2038bp
X = 7.0657 ng of insert

We wanted to find the amount of uL of insert we would need to use. We diluted the initial concentration 50x because we wanted a pipettable amount of insert. Since we knew we wanted the final amount of nanograms to be 7.0657, we calculated as shown below:

''DILUTION'': 555.48/50 = 11.012
''CALCULATION'': 11.012 ng/ 1uL = 7.065 ng/ x uL
x = .64 uL of insert

After we had done these calculations, half of the group started to carryout the ligation of the tRNA inserts into empty plasmids. We needed 7 ligations total: 6 were the tRNAs we needed, while the last one is a negative control. These are the final amounts of what we used:

[[Image: Trnaligation.png]]

The other half of the group (which included me) prepared the agarose gels so that we could run gels for the digested reporters and the PCR inserts. We calculated that the digested reporters needed an agarose of 4% and the PCR reporters needed an agarose of 1.45%. We allowed this gel to form for about 45 minutes to an hour. We prepared both the gels and allowed them to run through lunch.
After lunch, we took pictures of our gels and noticed that the third lane in the PCR gel (third lane from the top in the bottom photo) did not develop a band. The lane was supposed to contain the CUAGU insert for Tetracycline Resistance.

[[Image: 2009-06-12 15hr 54min.jpg]] [[Image:2009-06-12_16hr_07min.jpg]]

Romina Clemente and I decided to repeat this PCR using the TetResistance and RBS+TetResistance plasmids as templates. We labeled J31007 as "2*" and S03710 as "2!". We had to recalculate how much template DNA to use. We let the PCR run overnight. We also verified that the digestion fragments (both the plasmid and the insert) on the other gel were the correct size. The plasmids, which are the brighter fragment in the gel with only 2 lanes, contain the plasmid base pairs along with the rest of our reporter genes. We isolated these plasmids from the gel today and stored them for purification on Monday.

In the meantime, other members of our lab had finished the ligation of the tRNA genes to the empty plasmids and were now transforming these into competent cells. They placed the agar plate with these cells in the fridge overnight.

We also observed the transformations that we had performed yesterday and noticed that I13453 did not form any colonies. J04500 and J3102 did form colonies.

To Do:
1. PCR: Dig, Ligate, Transform
Clean and concentration column, digest, clean and concentration column, ligate.
2. Gel purification of our digestion plasmids
3. Check if colonies have grown for our tRNAs

==Saturday, June 13, 2009==

Alyndria Thompson came in on Saturday morning to remove the tRNA plates and put the PCR reactions in the fridge.

'''tRNA Colonies'''

She counted the colonies of tRNA on each plate.

[[Image:TRNA_colonies_6_13_09.png]]

She placed the plates in the large fridge on the top shelf at 9:23 am. She talked to Dr. Campbell who said we will have to try to "work them up".

'''PCR'''

Products were placed in orange rack of smaller fridge. [2*, 2!]

==Sunday, June 14, 2009==

We decided to re-ligate and re-transform all of the tRNA inserts because we did not get very good yields of colonies from the transformations.

We also stored liquid cultures of the colonies that greater than a 2:1 variable:negative control ratio just in case we wanted to use them for mini preps in the future.

We don't have usable sterile small pipet tips to use for the ligation of tRNA inserts so we are only pipetting the vectors into the tubes tonight. We are filling the empty pipet tip boxes with pipet tips. We put these in the Autoclave to be sterilized.

IGEM Notebook

2009-06-15T00:44:03Z

ShPunjabi: /* Friday, June 12, 2009 */

==Wednesday, June 3, 2009==

Romina Clemente and I are trying to find suitable reporter proteins to use. Yesterday, Leland Taylor and Alyndria Thompson were working on ways to insert the gene sequences into the plasmid. Upon seeing how they wanted to manipulated the reporter gene to include the logical clauses, we came up with a few criteria for the reporter genes we would use. The following criteria for genes are listed in order of the broadest aspect to look at to the narrowest aspect:
a) Doesn't contain restriction sites for the 4 restriction enzymes (EcoR1, Xbal, Spel, Pst1) used to cleave the Biobrick part out of the plasmid.
b) Contains 6 cutter restriction sites.
c) These restriction enzymes aren't blunt (cleave straight down at one spot).
d) These restriction sites are close to thge 5' (beginning) end of the sequence.
e) These enzymes are easiest to work with and cheapest.
We are finding the part numbers of the reporter genes we want to use (antibiotic resistance, fluorescence, LacZ) through our own GCAT because we know these ones work. We are then locating these parts on the parts registry [1] website. We copied and pasted the gene sequences we obtained from the registry onto the ApE software [2]. From here, we were able to generate a genetic map of each gene that outlined each restriction site that fit our criteria. We put each genetic map, alongside the part number used, into a Word document.

http://gcat.davidson.edu/GcatWiki/images/0/0e/Restriction_Site_Mapping_on_Reporter_Genes.doc

Later on in the day, we decided that the best way to test for suppression would involve placing the 5mers at the beginning of the reporter gene instead of inserting them into the reporter gene. To do this, we decided that the gene along with its start codon need to be expressed after the 5mer. We will insert a BioBrick plasmid with a 5mer and start codon that will be incorporated before the gene:
RBS-6-8nt-ATG-5mer--ATG-gene

Leland Taylor assembled the oligos that we will need for the BioBrick pieces.

In order to test whether the reporter protein would be expressed regardless of the 5mer being suppressed or not, we would need to remove the suppressor tRNAs from the cell.

Meanwhile, I helped figure out the coding sequence that we will need for the suppressor tRNAs that will bind to the 5mers. We decided that we would be using the following suppressor tRNAs on the Davidson side: CUAGU, CCCUC, CGGUC, CCAUC, and CCACU. We searched through several papers, with little result and finally emailed Dr. Christopher Anderson of UC Berkeley to request the DNA sequence. He emailed us back with a generic DNA code that we could use to create the tRNA; however, for each suppressor tRNA we would use, we would need to change base pairs in the anticodon loop.

The length of the tRNA is 92 nt. The anticodon loop is comprised of 9 nt. Once we have BioBricked the DNA sequence with 4 restriction sites and supplemental nucleotides, the length of the entire gene is 144 nucleotides. This is made up of the 92 nt +22 nt on each side of the sequence (44) + 4 nt on each side of the restriction ends (8).

At the end of the day, we decided to use tetracycline resistance and RFP as our reporter proteins for this tester experiment.

==Thursday, June 4, 2009==

This morning, I began the day by learning how to use the PEARL prgram. Olivia Ho-Shing sat with me and walked me through the program script. And then, Leland Taylor and Shashank Suresh had a problem they wanted us to solve. Leland explained that we need to make sure there is a stop codon in the gene if we fail to suppress the 5mer. The stop codon needs to be before we find another 5mer. They also wanted us to run through both the RFP and Tet Resistance genes to make sure that they did not contain any of the anticodon sequences from our 5 frameshift suppressor tRNAs so as to throw the translation out of frame. They further wanted us to confirm that if we do have successful suppression, there are no stop codons in the middle of the RFP and Tet Resistance genes. PEARL did not find any matches for the suppressor tRNAs within genes. We found 3 matches for stop codons UAA. Two of these occured at the end of the Tet and one at the end of RFP, where they should normally be to end translation into a protein.

Following this, Olivia Ho-Shing and I tried to determine how we can add BioBrick ends to our single stranded DNA sequences that code for the suppressor tRNAs. Our ultimate goal is to find complements for these single strands so they can be put into plasmids. We found the standard prefixes and suffixes for sequences that do not contain ATG (becuase this sequence is a functional RNA and will not be translated) and we placed these before and after the altered 92bp sequence that Dr. Anderson provided us with. The prefixes and suffixes are necessary because they are the extra nucleotides that will be mimicking a "restriction enzyme cutting site". The single strand appeared as below after the Bio Brick prefixes and suffixes were added:

22ntPREFIX--32nt--VARIABLE ANTICODON LOOP--51nt--21ntSUFFIX

We used the suppressor codon CUAGU as our example and placed its appropriate anticodon loop in the sequence. We put this into the lancelator that would give us the other strand as well as the oligos we would need for ideal construction of this double stranded DNA fragment to put into the plasmid. We recieved a total of 4 oligos, 2 of which were variable and 2 of which were constant for all 6 different suppressor tRNAs. Using this double strand, we simulated a restriction enzyme digestion by EcoRI and PstI. We took off the G at both ends of the top strand and then removed the first five nucleotides on each end of the bottom strand. This made the top strand longer than the bottom one. We then proceeded to take the two variable strands and changed the anticodon loop nt for each suppressor tRNA we used.

Around lunchtime, we encountered a problem with the BioBrick scar in the "ATG-5mer subpiece" of the test project. It turns out that after annealing the sticky ends of the Xba1 and Spe1 sites, the scar created had a stop codon TAG (UAG) that was in frame with the suppressed codon. When the Xba1 site on insert anneals to the Spe1 site on the plasmid (the green portions), we get a scar that reads TACTAG.
[[Image:screen-capture.png]]

For the afternoon, Olivia Ho-Shing and I finished assembly of the oligos of the suppressor tRNA codes. We assembled the document below:

http://gcat.davidson.edu/GcatWiki/images/5/5e/TRNAoligostoOrder.doc

We were assigned tasks of creating "cartoons" to illustrate the phenomenon that had occurred. No one could successfully do this however, because we could not understand how the piece had formed. The parts registry website provided wrong information on the restriction site of Spe1 which slowed our efficiency. This project turned into an effort to try to find two other different restriction enzymes that have complementary sticky ends other than Xba1 and Spe1. We want to get rid oc the scar that contains TACTAG.

We ended up deciding that a hybrid of two ideas was needed: restriction enzymes would need to be used to make several reporter gene sequences with different beginnings dependending on the suppressor tRNA sequence needed.

==Friday, June 5, 2009==

Our goal for this morning was to discuss the hybrid PCR approach to assemble the part that comes before the gene we wanted to express. We decided that we would have a BioBrick prefix and a 5mer followed by a sequence of nucleotides whose identity was up to us. Using PCR we would get the second double strand if we have the following:

BioBrick Prefix--ATG--5mer--gene until we get to restriction site

The PCR primer would attach to the ATG and the 5mer and would replicate the complementary base pairs to make a double stranded DNA sequence. As several of us were confused about PCR methods, we looked at the online link provided by Alyndria Thompson's link to the PCR interactive website which was very helpful. We also did some research to discover that the restriction enzyme BseRI (which is contained in the YFP that we wanted to alter) is not compatible with any other restriction enzyme. This is good because we did not want to plasmid containing this gene to close up with Xba1 once we digested it.

The team experienced some confusion with the hybrid idea and spoke to Clif Davis of Missouri Western University on the phone. He told us that there will be one part that is a fragment of the beginning of some nucleotides as a restriction cleaving site, ATG, the logical clause, and the start of the YFP gene that will be placed into a plasmid containing a space. To the left of the space will be the BioBrick Prefix and the right of the space will contain the rest of the genes in the YFP. Therefore, we decided not to alter any BioBrick ends and just look at restriction sites at the beginning of reporter proteins. The small fragment that will be placed into the plasmid will be made by PCR. The PCR will attach a primer onto the ATG and 5mer and replicate.

While we were discussing this, we expanded upon the idea that we can do whatever we want with the "suffix" portion between the 5mer and the beginning of YFP. We decided it would be best to remove ATG and add the first 5 amino acids and the restriction enzyme site. Along with this, if we have the 5mer coding for a certain amino acid (ie methionine) we could replace another codon that codes for methionine WITH that 5mer so long as this codon is contained within the first 15 nt. This way, we would not be altering the protein.

Olivia Ho-Shing came up with an idea of cascading PCR effect for longer logical clauses. Post translational modification of the protein was another one of her ideas. This applies because although we may code for the protein in a given way, the protein may modify or fix itself up to perform a slightly different function in its tertiary form.

Dr. Campbell suggested that we can look up restriction enzymes 400-500 base pairs into the reporter protein we would like to use; however, we have more flexibility in our procedure if we choose more "hardy" cutters. So instead, we could cut with EcoRI instead of with Xba1.

During the afternoon, we set about three tasks:
1) Which primers to use
2) Which promoters to use
3) Which restriction enzymes and how they will determine use of reporter proteins

Olivia Ho-Shing and I began looking up which restriction enzymes were strong so we could base our reporters on that. We began with Tet and RFP since those were the two reporters we had started out with choosing. I found a website that provided enzyme cleavage efficiency based upon the proximity of the site to the end of the PCR fragment that contains it. We discovered the following based upon the attached documents:

http://gcat.davidson.edu/GcatWiki/images/8/8f/Restriction_Enzyme_Cleavage_Efficiency.doc
http://gcat.davidson.edu/GcatWiki/images/9/9a/07Jun09_RE_Cutting_Efficiency_oh.doc

We decided that these two reporters contained efficient enzymes in a good quantity to choose from. On Monday, we will look through these enzymes once more and finalize which enzymes we would like to use with RFP and Tet.

==Sunday, June 7, 2009==

We did preparations for the mini prep this evening. The following notes are courtesy of Olivia Ho-Shing.

1. Sterically transfer ~2mL LB+AMP (AMP are ampicillin resistant plasmids) *Never put fluid back into bottle once it has entered pipette*
2. Use forceps to grab toothpick, scrap some frozen cells, rub in tube with top

'''Tubes'''

S03511 (2)

K091111

B0030

I715039

K091112

I715039

J31007 (2)

B0034

E1010

S03710

==Monday, June 8, 2009==

We reviewed the PCR process this morning as well as the restriction enzyme cleavage process.

Then, Leland Taylor, Olivia Ho-Shing and I all worked on converting the tRNA oligos we made to the template that Missouri Western used. The oligos we had previously made were different from the ones that Dr. Anderson had originally emailed us because further communication between Dr. Anderson and Missouri Western resulted in a better template.

We tried several times to minimize the number of oligos while keeping their individual lengths under 80bp. We managed to do this in the end by creating 7 oligos, 2 of which are variable between the suppressor tRNAs.
We reviewed the following document and finalized the oligos that we needed to order for the frameshift suppressor tRNAs. We decided to keep both the 10bp and 9bp anticodons of CCAUC.

I also looked up the amino acids that corresponded to the FS (frameshift suppressor) tRNAs for future use:

1. CUAGU = Leu
2. CCCUC = Pro
3. CGGUC = Arg
4. CCACU = Pro
5. CCAUC* = Pro
*9 and 10 bp anticodons

Also, we have decided to use NcoI as the restriction enzyme for RFP. This cleavage site occurs 419 bp into the gene. We have decided to use BamHI as the enzyme for Tetracycline Resistance and the cleavage site occurs 290bp into the gene. Both of these enzymes are very powerful and hardy.

We also looked over Missouri Western's tRNA oligos and primer oligos which we approved.
Our oligos were also approved by Missouri Western and Dr. Campbell ordered them while we supervised.

In the afternoon we did the miniprep. The mini prep directions were as appear in the link below.

http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_MiniPrep.html

We then worked with the Nano Prep to dilute our solution of DNA to an absorption of 1.5 and then we made labels for the tubes. The chart below shows relevant information we obtained from the NanoDrop for each of the reporter plasmids.

[[Image: Screen-capture-6.png]]

==Tuesday, June 9, 2009==

'''Preparations for restriction enzymes digestion of plasmids'''

Purpose: To determine the size of the plasmid inserts and ensure that plasmids are what we extracted from the cells during the mini-prep.

Things we need:
DNA ~3uL
Buffer 10x (Want to dilute to 1x) so add 2uL
Enzyme 1 ~1uL
Enzme 2 ~1uL
Water ~13uL
Final Volunter about 20 uL.

Need to choose the right buffer for the restriction enzymes. All buffers come in 10x (10 times more concentrated than what you want it to be when you use it). [http://gcat.davidson.edu/GcatWiki/index.php/Davidson_Missouri_W/Double_Digest_Guide Buffer Website]

Total volume of enzymes has to be 10% or less than final volume (most volume 2uL). Barely submerge the pipet tip into the solution when pipeting the enzymes.
Star (*) activity: Restriction enzyme stops cutting specifically and starts cutting everywhere.

DNA can't be seen by itself so we add EtBr (the bigger the insert, the more will bind) so bigger strands appear brighter. samples.

The order of entry doesn't matter, but make sure to enter the enzymes last.

Will have lots of labeled tubes and will put 3uL of appropriate plasmids into the tubes.
In the meantime, we'll make a "Master Mix" of all the other ingredients in a bigger tube. Always make enough volume for one extra tube.

Enzymes usually have an optimal temperature of 37C. We will let this reaction go for an hour and then we will run it on a gel.

'''Gel Electrophoresis Protocol'''

Website online for ideal gel concentration for resolving different sizes of molecules [http://gcat.davidson.edu/iGEM08/gelwebsite/gelwebsite.html Gel Concentration Website].
See protocol on the Davidson/Missouri Western Wookie for running molecules in the gel and how to digest with restriction enzymes.

'''What's Next?'''

1. 5' end additions
a. Using PCR
b. Cut at the restriction sites EcoR1 and either Bam H1 or Nco1
c. Cut plasmid and reporter with same restriction enzymes as above
d. Run a gel and purify "keeper" DNA (Ligate transform, screen, sequence)
e. Then, we take the "keeper" DNA and combine it with the insert from part

We can have steps d and e ready in advance.

2. tRNAs
a. Assemble the oligos into genes (Requires lots of calculations beforehand, but the actual steps )
b. Plasmids cut up with EcoR1 and Pst1 '''WE CAN USE THE "WASTE" PLASMID FROM THE RESTRICTION ENZYME DIGEST'''
c. Gel purify the plasmids (Ligate transform, screen, sequence)

The following tables include Master Mix measurements, agarose gel concentrations and buffers.
http://gcat.davidson.edu/GcatWiki/images/0/08/Shamitapic3.png
http://gcat.davidson.edu/GcatWiki/images/d/d1/Shamitapic4.png
http://gcat.davidson.edu/GcatWiki/images/6/6f/Shamitapic5.png

We made the Master Mix with our DNA and put it in the incubator at 10:40 AM.

'''Preparing the Gels'''

Now, we will be making 2 gels, 1 at 0.8% and 2.5% (weight to volume percent). The gel volume is 60mL.

'''Definition of 1% solution:
1 gram of agarose/ 100mL buffer'''

So for a 0.8% solution: 0.8 grams agarose/100mL buffer AND we need 60mL (fixed volume for casts run) so multiply that by 0.6.
Final concentration:

.48 grams agarose/60mL buffer

1 uL of EtBr stock for 60mL of gel.
EtBr is in the buffer so it is constantly replenishing itself even though it is moving toward one end in order to make bands on the other end visible.

After Dr. Campbell's demonstration, we each helped prepare a 0.8% buffer solution using the agarose powder, buffer and EtBr. We mixed together the first two substances in a 200mL Erlenmeyer flask and heated it for 120 seconds in the microwave. We then added EtBr and poured the gel solution into the gel cast.

'''Agar Plate'''

After lunch, we grew some more cells for each of the 11 parts in an agar plate labeled "LBAMP-AMC-9 June" so that so would have extra cells that could be stored for up to 2 weeks.
We made small circles on the agar plate and numbered them 1-11. The numbers corresponded to the following parts. We put the plate in the incubator overnight to let the cells grow.

1. B0030 AMC
2. K091111
3. S03710
4. I715039 OEH
5. S03511 ORH
6. I715039 AST
7. B0030 SDP
8. K091112
9. J31007 LJH
10. J31007 REC
11. S03511

'''Unloading the Gels'''

The following shows which parts we entered in which gels:

[[Image: screen-capture-2.png]]

We unloaded the 2.5% agarose first and after observing the bands under a UV light, put the gel into the BioRad machine to observe the bands. The image below is of a Molecular Weight Marker that shows the number of bp in control fragments of DNA according to the distance they travel down the gel.

[[Image: DNA_molecular_weight_marker_gel.png]]

We obtained the following printout for the first gel that was 2.5% Agarose.
[[Image: 2009-06-09_14hr_44min.jpg]]

The bands in the first well at the bottom are those of the molecular weight marker. We skipped the lane above (to the right) of the MWM and entered the rest of the inserts as described in the above table. A light band is visible for each of the 4 other wells. In Lanes 3 and 4 we concluded we are supposed to have approximately 45 bp because the insert is 15bp + 30 bp on either side of the insert (Xba1 and Spe1 restrictions sites + sticky ends + extra bases). We used the same logic for Lanes 5 and 6 which are supposed to contain approximately 86 base pairs.

One discrepancy in this gel, however, is that Lanes 5 and 6, which according to their labels respectively contain the LacIQ promoter and the Lac IQ1 promoter. According to the information online at the parts registry website, pLacIQ1 should be the same length as pLacIQ. The gels do not show this, but instead show that the sample in Lane 5 is smaller than that in Lane 6 because Lane 5 traveled further. Leland Taylor and Romina Clemente sent out an email to Pallavi Penumetcha to ask about this because she had written a paper about this.

We then unloaded the 0.8% agarose gel and then placed this in the BioRad machine under the same protocol as above. The following image is the 0.8% Agarose.

[[Image:2009-06-09_15hr_21min.jpg]]

We observed that the bands in Lane 1 (which is now at the top) are examples of high intensity bands. The plasmids contained in the original well are twice the intensity of the insert.

In Lane 5, however, we observed several different bands. This could be due to the extra DNA that was sitting on the top of the well. We decided to re-expose the gel to get better observations on Lanes 3 and 4 because the bands were not as intense. We anticipated seeing a band at about 250 bp because this was the size of the insert (plus extra bp). After re-exposing the gel, we did not find any difference and still did not see any bands in these lanes. We have decided to redo the gel on the part S03511 and so we prepared the master mix with that DNA and incubated it so that we could run the gel tomorrow.

We also cut out the "empty" plasmids from the gel so that we could use this as vectors for the coding sequence of the suppressor tRNAs.

Tomorrow we will be planning out specific tasks for people once the oligos arrive.

==Wednesday, June 10, 2009==

We began this morning by making the agarose gel with the same protocol as yesterday. This time, since we are just re-doing both S03511 inserts, we will have a 1.8% agarose gel.

'''OUTLINE: WHERE DO WE STAND?'''

Yesterday
a. Isolated empty plasmids
b. Verified most inserts
c. Waiting for:
>tRNA oligos
>Primers
a. Will amplify the reporter gene in the plasmid itself

Next?
a. When Primers Arrive
> Amplify the reporter genes with primers (PCR takes ~3 hours)
> Calculate how much reporter plasmid we will need (Start with 50x more than we actually will need)
> Cut amplified sequence (EcoR1 and BamH1/Nco1) then purify what we need
> Cut reporter plasmid (EcoR1 and BamH1/Nco1) then purify what we need
> Ligate amplified sequence and reporter plasmid
b. When tRNA oligos arrive
> Calculate how much of each oligo we will need (takes a while...)
> "Boil and cool" tRNA oligos together
> Ligate fragments (dsDNA) into plasmids we isolated yesterday (~5 minutes)
> Transform the plasmids (~20 minutes)

* LOOK THROUGH LIGATION PROTOCOL (How to assemble genes)
* LOOK THROUGH TRANSFORMATION PROTOCOL

''What we need to get done today'':
1. Prepare the control for the Tet Resistance by BioBricking the part pLac+RBS (S03511) with the TetA (J31007).
2. Check the part number for RFP and also for pLac+RBS+RFP
3. Read through ligation protocol
4. See how many nanograms of plasmid we will need for the tRNAs and for the inserts and multiply this number by 50.
> Check if we have enough plasmids from our mini-prep
5. Purify the plasmids from the gel

After we made this outline, Leland, Olivia and I analyzed the gel we ran this morning and did not see a more than one band in the 2 lanes that contained DNA from S03511 (image below). Since we did not see multiple bands in yesterday's gel either, we conclude that there is something wrong with the DNA rather than with the restriction enzymes that we used to cut it. We concluded that there is probably not an insert in this plasmid both the part and the plasmid are useless.

[[Image: 2009-06-10 10hr 30min.jpg]]

Now, we do not have an immediately accessible promoter. We have decided to order the pBAD promoter from Missouri Western and we will attach the RBS+Tet to this plasmid. Our insert for this will be the larger gene because it is easier to manipulate in the gel than the small gene.

Alyndria Thompson and Romina Clemente are figuring out the size of the plasmids including the reporter genes.

After lunch we performed several steps to isolate and purify the gel to get the empty plasmids will be the vectors for the tRNA DNA. Then, we used the NanoDrop to assess the purity of our DNA. We found that the plasmids were more pure than we had expected. The following values were obtained:

Abs 1.850
A-260 10 mm path 0.172
A-280 10 mm path 0.075
260/280 2.28
260/230 0.09

We then followed the protocol for preparing the agar plates. We put the plate with the agar solution in the Autoclave.

While the solution was in the autoclave, we discussed that we don't really need a promoter in front of RBS+Tet for the gene to be expressed because Tet expression is so strong that the gene would translate without a promoter. However, it would be a good idea to have the promoter because if it was possible to reinforce the translation of the reporter protein, we wanted to do that.

We found that there were some tubes in the lab fridge that contained the pBAD and pLac promoters with RBS. We decided to use these as the promoter and RBS for our experiments. The pLac promoter will be used for RFP and the pBad promoter used with Tet Resistance. We decided to use the pBad with Tet because it is weaker in comparison to pLac. As expression of the Tet gene can be toxic to a cell, we do not want to overexpress the gene with a stronger promoter.

Once the agar solution was sterilized in the Autoclave, we removed it, cooled it, and pipetted the antibiotic resistance into it. We then poured the solution into the plates, removed any bubbles, and let the gel form.

Alyndria Thompson and Romina Clemente concluded that we had more than enough recieving vectors for both the RFP and Tetracycline genes. Since we need 50 nanograms, it is wise to have at least 50 times that much that we can work with, so we need at least 2,500 ng.

RFP 1: 77.6 x 4 = 310.4 ng/uL
x 20 uL = 3,272 ng
RFP 2: 59.5 x 4 = 238 ng/uL
x 20 uL = 4,760 ng
Tet: 40.9 x 4 = 163.6 ng/uL
x 20 uL = 3,272 ng

According to these calculations we have more than 50 times what we need for both instances of RFP and Tet.

Tomorrow, we can prepare the Master Mix for the digestions of both reporter plamids and then add the restriction enzymes once they arrive. Then, we can follow through with the rest of the plans we had made above in the section titles "Next?".

==Thursday, June 11, 2009==

We began this morning by calculating how many uL we would need for each of the mixes for digesting our reporter plasmids. We decided to double the final volume of each mix to 40uL instead of 20uL. Since we knew the concentration of each plasmid and also knew we needed 2500 ng, we cross multiplied to find the uL of DNA we would need for each.

Concentration Calculations:
310.4 ng/1 uL = 2500ng/x uL
x = 8.05 uL

Here is a chart indicating how much of each we used for the 2 mixes.

[[Image: DoubledigestRFPTetR.png]]

On the Promega website, we found a list of compatible buffers for double digestions. Buffers E and H are compatible for the Tet plasmid and RFP plasmid digestions respectively. We fortunately have both buffers in stock.

[[Image:BamEcoR1.png]] [[Image:NcoEcoR1.png]]

We waited for our waited for the restriction enzymes to arrive and in the meantime, we transformed the plasmids with pLac+RBS, pBad, and pBad+RBS into ''E.coli'' cells. We want the ''E.coli'' cells to replicate the plasmids with these genes because we have a limited amount of genes. [http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_Transformation.html Zippy Plasmid Transformation]

Once our oligos and restriction enzymes arrived, we got to work right away!

'''PCR (Polymerase Chain Reaction)'''

We calculated how much plasmid we will need for the PCR. We decided to dilute the concentration of our plasmids a hundred fold so that we could get pipetable amounts.

''Change in Concentrations''
310.4 ng/uL x 1/100 (dilution factor) = 3.104 ng/uL (RFP)
163.6 ng/uL x 1/100 (dilution factor) = 0.164 ng/uL (Tet)

''Example shown with RFP''
3.104 ng/1 uL = 1ng/ x uL, x = .32
For Tet, x = .61

We also calculated how much of each primer we would need with the following equation:

C1V1 = C2V2
(100 uM) (x) = (1 uM)( 100 uL)
x = 1 uL

[[Image: screen-capture-8.png]]

Each of the 5 of us made had 2 tubes (one for each reporter protein) because there were 5 different forward primers.To each tube, we added the template DNA (either RFP or Tet), water, the respective reverse primer (either RFP or Tet), and the forward primer depending on which 5mer we had chosen. My 5mer was CCAUC. Finally, we added the Master Mix last because this contained the enzyme (DNA polymerase) that would amplify our desired sequence.

[[Image: Pcrtubes.png]]

We followed the temperature cycles so that PCR could occur. The annealing temperature needed to be 5C less than the smallest melting temperature of the primers. Since the reverse primer of RFP had a melting temp of 66C, we set the temperature of the 3rd cycle on 61C.

[[Image: Temperaturecycle.png]]

'''tRNA Oligo Assembly'''

We needed to calculate the concentration of each of the oligo before carrying out the assembly. The stock concentration of each of the oligo was 100 uM. Since we wanted to have 5uM of the oligo in the test tube, and we know that final volume of the test tube is 20 uL, we just cross multiplied to find the amount we needed to add to the tubes to get this concentration.

(100uM)x = (5uM)(20uL)
x = 1 uL

We added 1 uL of each oligo to the tubes.

We had six total tRNAs we needed to assemble so each of us took one FML (one person took two) and to each tube we added 1uL of each oligo for a total of 7uL, 2uL of buffer, and 11uL of water for a total volume of 20 uL. Five of the oligos were constant and 2 varied for each of the different tRNAs. So everyone pipetted the same 5 oligos and everyone also had 2 unique oligos to add. When all of the oligos were added and the tubes ready, we boiled the tubes at 50C for 5 minutes. We then allowed the tubes to remain in the water to cool very slowly overnight so that the annealing process could take place.

The last thing we did at the end of the day was finish off our plasmid digestion that we had begun in the morning. We added the buffer, DNA, water, and restriction enzymes and placed this in the incubator for overnight digestion.

'''To Do''':

1. Isolate our desired dsDNA from the PCR.
2. Verify our tRNA inserts and eventually transform these into plasmids.
3. Check our agar plates to see colonies that have replicated our pLac+RBS, pBAD+RBS, and pBAD genes.

==Friday, June 12, 2009==

This morning, we began by outlining the main items on today's agenda.

1. tRNAs: Ligation, Transformation
2. Digestion reporters (run all of it and gel purify)
3. Transformed cells
4. PCR: Gel run an aliquot to verify (5uL)

We used the equation given by the Ligation protocol to calculate how many nanograms of insert we would need. Initially, we started out calculated the MW average of the 9 and 10 anticodon loops of tRNAs which were 15828.23 ug/umols and 159016.6 ug/umols respectively. We then saw, however, that these would not be helpful values in the process. Instead we started out with the equation below to find the final ng of insert we would need for the ligation.

[[Image: ligationcalculations.png]]

We calculated our initial concentration in ng/uL of insert as shown below.

[[Image: ligationequation.png]]
X nanograms of insert = [(2)(144bp)(50 ng linearalized plasmid)]/2038bp
X = 7.0657 ng of insert

We wanted to find the amount of uL of insert we would need to use. We diluted the initial concentration 50x because we wanted a pipettable amount of insert. Since we knew we wanted the final amount of nanograms to be 7.0657, we calculated as shown below:

''DILUTION'': 555.48/50 = 11.012
''CALCULATION'': 11.012 ng/ 1uL = 7.065 ng/ x uL
x = .64 uL of insert

After we had done these calculations, half of the group started to carryout the ligation of the tRNA inserts into empty plasmids. We needed 7 ligations total: 6 were the tRNAs we needed, while the last one is a negative control. These are the final amounts of what we used:

[[Image: Trnaligation.png]]

The other half of the group (which included me) prepared the agarose gels so that we could run gels for the digested reporters and the PCR inserts. We calculated that the digested reporters needed an agarose of 4% and the PCR reporters needed an agarose of 1.45%. We allowed this gel to form for about 45 minutes to an hour. We prepared both the gels and allowed them to run through lunch.
After lunch, we took pictures of our gels and noticed that the third lane in the PCR gel (third lane from the top in the bottom photo) did not develop a band. The lane was supposed to contain the CUAGU insert for Tetracycline Resistance.

[[Image: 2009-06-12 15hr 54min.jpg]] [[Image:2009-06-12_16hr_07min.jpg]]

Romina Clemente and I decided to repeat this PCR using the TetResistance and RBS+TetResistance plasmids as templates. We labeled J31007 as "2*" and S03710 as "2!". We had to recalculate how much template DNA to use. We let the PCR run overnight. We also verified that the digestion fragments (both the plasmid and the insert) on the other gel were the correct size. The plasmids, which are the brighter fragment in the gel with only 2 lanes, contain the plasmid base pairs along with the rest of our reporter genes. We isolated these plasmids from the gel today and stored them for purification on Monday.

In the meantime, other members of our lab had finished the ligation of the tRNA genes to the empty plasmids and were now transforming these into competent cells. They placed the agar plate with these cells in the fridge overnight.

We also observed the transformations that we had performed yesterday and noticed that I13453 did not form any colonies. J04500 and J3102 did form colonies.

To Do:
1. PCR: Dig, Ligate, Transform
Clean and concentration column, digest, clean and concentration column, ligate.
2. Gel purification of our digestion plasmids
3. Check if colonies have grown for our tRNAs

==Saturday, June 13, 2009==

Alyndria Thompson came in on Saturday morning to remove the tRNA plates and put the PCR reactions in the fridge.

'''tRNA Colonies'''

She counted the colonies of tRNA on each plate.

[[Image:TRNA_colonies_6_13_09.png]]

She placed the plates in the large fridge on the top shelf at 9:23 am. She talked to Dr. Campbell who said we will have to try to "work them up".

'''PCR'''

Products were placed in orange rack of smaller fridge. [2*, 2!]

IGEM Notebook

2009-06-15T00:41:32Z

ShPunjabi: /* Saturday, June 13, 2009 */

==Wednesday, June 3, 2009==

Romina Clemente and I are trying to find suitable reporter proteins to use. Yesterday, Leland Taylor and Alyndria Thompson were working on ways to insert the gene sequences into the plasmid. Upon seeing how they wanted to manipulated the reporter gene to include the logical clauses, we came up with a few criteria for the reporter genes we would use. The following criteria for genes are listed in order of the broadest aspect to look at to the narrowest aspect:
a) Doesn't contain restriction sites for the 4 restriction enzymes (EcoR1, Xbal, Spel, Pst1) used to cleave the Biobrick part out of the plasmid.
b) Contains 6 cutter restriction sites.
c) These restriction enzymes aren't blunt (cleave straight down at one spot).
d) These restriction sites are close to thge 5' (beginning) end of the sequence.
e) These enzymes are easiest to work with and cheapest.
We are finding the part numbers of the reporter genes we want to use (antibiotic resistance, fluorescence, LacZ) through our own GCAT because we know these ones work. We are then locating these parts on the parts registry [1] website. We copied and pasted the gene sequences we obtained from the registry onto the ApE software [2]. From here, we were able to generate a genetic map of each gene that outlined each restriction site that fit our criteria. We put each genetic map, alongside the part number used, into a Word document.

http://gcat.davidson.edu/GcatWiki/images/0/0e/Restriction_Site_Mapping_on_Reporter_Genes.doc

Later on in the day, we decided that the best way to test for suppression would involve placing the 5mers at the beginning of the reporter gene instead of inserting them into the reporter gene. To do this, we decided that the gene along with its start codon need to be expressed after the 5mer. We will insert a BioBrick plasmid with a 5mer and start codon that will be incorporated before the gene:
RBS-6-8nt-ATG-5mer--ATG-gene

Leland Taylor assembled the oligos that we will need for the BioBrick pieces.

In order to test whether the reporter protein would be expressed regardless of the 5mer being suppressed or not, we would need to remove the suppressor tRNAs from the cell.

Meanwhile, I helped figure out the coding sequence that we will need for the suppressor tRNAs that will bind to the 5mers. We decided that we would be using the following suppressor tRNAs on the Davidson side: CUAGU, CCCUC, CGGUC, CCAUC, and CCACU. We searched through several papers, with little result and finally emailed Dr. Christopher Anderson of UC Berkeley to request the DNA sequence. He emailed us back with a generic DNA code that we could use to create the tRNA; however, for each suppressor tRNA we would use, we would need to change base pairs in the anticodon loop.

The length of the tRNA is 92 nt. The anticodon loop is comprised of 9 nt. Once we have BioBricked the DNA sequence with 4 restriction sites and supplemental nucleotides, the length of the entire gene is 144 nucleotides. This is made up of the 92 nt +22 nt on each side of the sequence (44) + 4 nt on each side of the restriction ends (8).

At the end of the day, we decided to use tetracycline resistance and RFP as our reporter proteins for this tester experiment.

==Thursday, June 4, 2009==

This morning, I began the day by learning how to use the PEARL prgram. Olivia Ho-Shing sat with me and walked me through the program script. And then, Leland Taylor and Shashank Suresh had a problem they wanted us to solve. Leland explained that we need to make sure there is a stop codon in the gene if we fail to suppress the 5mer. The stop codon needs to be before we find another 5mer. They also wanted us to run through both the RFP and Tet Resistance genes to make sure that they did not contain any of the anticodon sequences from our 5 frameshift suppressor tRNAs so as to throw the translation out of frame. They further wanted us to confirm that if we do have successful suppression, there are no stop codons in the middle of the RFP and Tet Resistance genes. PEARL did not find any matches for the suppressor tRNAs within genes. We found 3 matches for stop codons UAA. Two of these occured at the end of the Tet and one at the end of RFP, where they should normally be to end translation into a protein.

Following this, Olivia Ho-Shing and I tried to determine how we can add BioBrick ends to our single stranded DNA sequences that code for the suppressor tRNAs. Our ultimate goal is to find complements for these single strands so they can be put into plasmids. We found the standard prefixes and suffixes for sequences that do not contain ATG (becuase this sequence is a functional RNA and will not be translated) and we placed these before and after the altered 92bp sequence that Dr. Anderson provided us with. The prefixes and suffixes are necessary because they are the extra nucleotides that will be mimicking a "restriction enzyme cutting site". The single strand appeared as below after the Bio Brick prefixes and suffixes were added:

22ntPREFIX--32nt--VARIABLE ANTICODON LOOP--51nt--21ntSUFFIX

We used the suppressor codon CUAGU as our example and placed its appropriate anticodon loop in the sequence. We put this into the lancelator that would give us the other strand as well as the oligos we would need for ideal construction of this double stranded DNA fragment to put into the plasmid. We recieved a total of 4 oligos, 2 of which were variable and 2 of which were constant for all 6 different suppressor tRNAs. Using this double strand, we simulated a restriction enzyme digestion by EcoRI and PstI. We took off the G at both ends of the top strand and then removed the first five nucleotides on each end of the bottom strand. This made the top strand longer than the bottom one. We then proceeded to take the two variable strands and changed the anticodon loop nt for each suppressor tRNA we used.

Around lunchtime, we encountered a problem with the BioBrick scar in the "ATG-5mer subpiece" of the test project. It turns out that after annealing the sticky ends of the Xba1 and Spe1 sites, the scar created had a stop codon TAG (UAG) that was in frame with the suppressed codon. When the Xba1 site on insert anneals to the Spe1 site on the plasmid (the green portions), we get a scar that reads TACTAG.
[[Image:screen-capture.png]]

For the afternoon, Olivia Ho-Shing and I finished assembly of the oligos of the suppressor tRNA codes. We assembled the document below:

http://gcat.davidson.edu/GcatWiki/images/5/5e/TRNAoligostoOrder.doc

We were assigned tasks of creating "cartoons" to illustrate the phenomenon that had occurred. No one could successfully do this however, because we could not understand how the piece had formed. The parts registry website provided wrong information on the restriction site of Spe1 which slowed our efficiency. This project turned into an effort to try to find two other different restriction enzymes that have complementary sticky ends other than Xba1 and Spe1. We want to get rid oc the scar that contains TACTAG.

We ended up deciding that a hybrid of two ideas was needed: restriction enzymes would need to be used to make several reporter gene sequences with different beginnings dependending on the suppressor tRNA sequence needed.

==Friday, June 5, 2009==

Our goal for this morning was to discuss the hybrid PCR approach to assemble the part that comes before the gene we wanted to express. We decided that we would have a BioBrick prefix and a 5mer followed by a sequence of nucleotides whose identity was up to us. Using PCR we would get the second double strand if we have the following:

BioBrick Prefix--ATG--5mer--gene until we get to restriction site

The PCR primer would attach to the ATG and the 5mer and would replicate the complementary base pairs to make a double stranded DNA sequence. As several of us were confused about PCR methods, we looked at the online link provided by Alyndria Thompson's link to the PCR interactive website which was very helpful. We also did some research to discover that the restriction enzyme BseRI (which is contained in the YFP that we wanted to alter) is not compatible with any other restriction enzyme. This is good because we did not want to plasmid containing this gene to close up with Xba1 once we digested it.

The team experienced some confusion with the hybrid idea and spoke to Clif Davis of Missouri Western University on the phone. He told us that there will be one part that is a fragment of the beginning of some nucleotides as a restriction cleaving site, ATG, the logical clause, and the start of the YFP gene that will be placed into a plasmid containing a space. To the left of the space will be the BioBrick Prefix and the right of the space will contain the rest of the genes in the YFP. Therefore, we decided not to alter any BioBrick ends and just look at restriction sites at the beginning of reporter proteins. The small fragment that will be placed into the plasmid will be made by PCR. The PCR will attach a primer onto the ATG and 5mer and replicate.

While we were discussing this, we expanded upon the idea that we can do whatever we want with the "suffix" portion between the 5mer and the beginning of YFP. We decided it would be best to remove ATG and add the first 5 amino acids and the restriction enzyme site. Along with this, if we have the 5mer coding for a certain amino acid (ie methionine) we could replace another codon that codes for methionine WITH that 5mer so long as this codon is contained within the first 15 nt. This way, we would not be altering the protein.

Olivia Ho-Shing came up with an idea of cascading PCR effect for longer logical clauses. Post translational modification of the protein was another one of her ideas. This applies because although we may code for the protein in a given way, the protein may modify or fix itself up to perform a slightly different function in its tertiary form.

Dr. Campbell suggested that we can look up restriction enzymes 400-500 base pairs into the reporter protein we would like to use; however, we have more flexibility in our procedure if we choose more "hardy" cutters. So instead, we could cut with EcoRI instead of with Xba1.

During the afternoon, we set about three tasks:
1) Which primers to use
2) Which promoters to use
3) Which restriction enzymes and how they will determine use of reporter proteins

Olivia Ho-Shing and I began looking up which restriction enzymes were strong so we could base our reporters on that. We began with Tet and RFP since those were the two reporters we had started out with choosing. I found a website that provided enzyme cleavage efficiency based upon the proximity of the site to the end of the PCR fragment that contains it. We discovered the following based upon the attached documents:

http://gcat.davidson.edu/GcatWiki/images/8/8f/Restriction_Enzyme_Cleavage_Efficiency.doc
http://gcat.davidson.edu/GcatWiki/images/9/9a/07Jun09_RE_Cutting_Efficiency_oh.doc

We decided that these two reporters contained efficient enzymes in a good quantity to choose from. On Monday, we will look through these enzymes once more and finalize which enzymes we would like to use with RFP and Tet.

==Sunday, June 7, 2009==

We did preparations for the mini prep this evening. The following notes are courtesy of Olivia Ho-Shing.

1. Sterically transfer ~2mL LB+AMP (AMP are ampicillin resistant plasmids) *Never put fluid back into bottle once it has entered pipette*
2. Use forceps to grab toothpick, scrap some frozen cells, rub in tube with top

'''Tubes'''

S03511 (2)

K091111

B0030

I715039

K091112

I715039

J31007 (2)

B0034

E1010

S03710

==Monday, June 8, 2009==

We reviewed the PCR process this morning as well as the restriction enzyme cleavage process.

Then, Leland Taylor, Olivia Ho-Shing and I all worked on converting the tRNA oligos we made to the template that Missouri Western used. The oligos we had previously made were different from the ones that Dr. Anderson had originally emailed us because further communication between Dr. Anderson and Missouri Western resulted in a better template.

We tried several times to minimize the number of oligos while keeping their individual lengths under 80bp. We managed to do this in the end by creating 7 oligos, 2 of which are variable between the suppressor tRNAs.
We reviewed the following document and finalized the oligos that we needed to order for the frameshift suppressor tRNAs. We decided to keep both the 10bp and 9bp anticodons of CCAUC.

I also looked up the amino acids that corresponded to the FS (frameshift suppressor) tRNAs for future use:

1. CUAGU = Leu
2. CCCUC = Pro
3. CGGUC = Arg
4. CCACU = Pro
5. CCAUC* = Pro
*9 and 10 bp anticodons

Also, we have decided to use NcoI as the restriction enzyme for RFP. This cleavage site occurs 419 bp into the gene. We have decided to use BamHI as the enzyme for Tetracycline Resistance and the cleavage site occurs 290bp into the gene. Both of these enzymes are very powerful and hardy.

We also looked over Missouri Western's tRNA oligos and primer oligos which we approved.
Our oligos were also approved by Missouri Western and Dr. Campbell ordered them while we supervised.

In the afternoon we did the miniprep. The mini prep directions were as appear in the link below.

http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_MiniPrep.html

We then worked with the Nano Prep to dilute our solution of DNA to an absorption of 1.5 and then we made labels for the tubes. The chart below shows relevant information we obtained from the NanoDrop for each of the reporter plasmids.

[[Image: Screen-capture-6.png]]

==Tuesday, June 9, 2009==

'''Preparations for restriction enzymes digestion of plasmids'''

Purpose: To determine the size of the plasmid inserts and ensure that plasmids are what we extracted from the cells during the mini-prep.

Things we need:
DNA ~3uL
Buffer 10x (Want to dilute to 1x) so add 2uL
Enzyme 1 ~1uL
Enzme 2 ~1uL
Water ~13uL
Final Volunter about 20 uL.

Need to choose the right buffer for the restriction enzymes. All buffers come in 10x (10 times more concentrated than what you want it to be when you use it). [http://gcat.davidson.edu/GcatWiki/index.php/Davidson_Missouri_W/Double_Digest_Guide Buffer Website]

Total volume of enzymes has to be 10% or less than final volume (most volume 2uL). Barely submerge the pipet tip into the solution when pipeting the enzymes.
Star (*) activity: Restriction enzyme stops cutting specifically and starts cutting everywhere.

DNA can't be seen by itself so we add EtBr (the bigger the insert, the more will bind) so bigger strands appear brighter. samples.

The order of entry doesn't matter, but make sure to enter the enzymes last.

Will have lots of labeled tubes and will put 3uL of appropriate plasmids into the tubes.
In the meantime, we'll make a "Master Mix" of all the other ingredients in a bigger tube. Always make enough volume for one extra tube.

Enzymes usually have an optimal temperature of 37C. We will let this reaction go for an hour and then we will run it on a gel.

'''Gel Electrophoresis Protocol'''

Website online for ideal gel concentration for resolving different sizes of molecules [http://gcat.davidson.edu/iGEM08/gelwebsite/gelwebsite.html Gel Concentration Website].
See protocol on the Davidson/Missouri Western Wookie for running molecules in the gel and how to digest with restriction enzymes.

'''What's Next?'''

1. 5' end additions
a. Using PCR
b. Cut at the restriction sites EcoR1 and either Bam H1 or Nco1
c. Cut plasmid and reporter with same restriction enzymes as above
d. Run a gel and purify "keeper" DNA (Ligate transform, screen, sequence)
e. Then, we take the "keeper" DNA and combine it with the insert from part

We can have steps d and e ready in advance.

2. tRNAs
a. Assemble the oligos into genes (Requires lots of calculations beforehand, but the actual steps )
b. Plasmids cut up with EcoR1 and Pst1 '''WE CAN USE THE "WASTE" PLASMID FROM THE RESTRICTION ENZYME DIGEST'''
c. Gel purify the plasmids (Ligate transform, screen, sequence)

The following tables include Master Mix measurements, agarose gel concentrations and buffers.
http://gcat.davidson.edu/GcatWiki/images/0/08/Shamitapic3.png
http://gcat.davidson.edu/GcatWiki/images/d/d1/Shamitapic4.png
http://gcat.davidson.edu/GcatWiki/images/6/6f/Shamitapic5.png

We made the Master Mix with our DNA and put it in the incubator at 10:40 AM.

'''Preparing the Gels'''

Now, we will be making 2 gels, 1 at 0.8% and 2.5% (weight to volume percent). The gel volume is 60mL.

'''Definition of 1% solution:
1 gram of agarose/ 100mL buffer'''

So for a 0.8% solution: 0.8 grams agarose/100mL buffer AND we need 60mL (fixed volume for casts run) so multiply that by 0.6.
Final concentration:

.48 grams agarose/60mL buffer

1 uL of EtBr stock for 60mL of gel.
EtBr is in the buffer so it is constantly replenishing itself even though it is moving toward one end in order to make bands on the other end visible.

After Dr. Campbell's demonstration, we each helped prepare a 0.8% buffer solution using the agarose powder, buffer and EtBr. We mixed together the first two substances in a 200mL Erlenmeyer flask and heated it for 120 seconds in the microwave. We then added EtBr and poured the gel solution into the gel cast.

'''Agar Plate'''

After lunch, we grew some more cells for each of the 11 parts in an agar plate labeled "LBAMP-AMC-9 June" so that so would have extra cells that could be stored for up to 2 weeks.
We made small circles on the agar plate and numbered them 1-11. The numbers corresponded to the following parts. We put the plate in the incubator overnight to let the cells grow.

1. B0030 AMC
2. K091111
3. S03710
4. I715039 OEH
5. S03511 ORH
6. I715039 AST
7. B0030 SDP
8. K091112
9. J31007 LJH
10. J31007 REC
11. S03511

'''Unloading the Gels'''

The following shows which parts we entered in which gels:

[[Image: screen-capture-2.png]]

We unloaded the 2.5% agarose first and after observing the bands under a UV light, put the gel into the BioRad machine to observe the bands. The image below is of a Molecular Weight Marker that shows the number of bp in control fragments of DNA according to the distance they travel down the gel.

[[Image: DNA_molecular_weight_marker_gel.png]]

We obtained the following printout for the first gel that was 2.5% Agarose.
[[Image: 2009-06-09_14hr_44min.jpg]]

The bands in the first well at the bottom are those of the molecular weight marker. We skipped the lane above (to the right) of the MWM and entered the rest of the inserts as described in the above table. A light band is visible for each of the 4 other wells. In Lanes 3 and 4 we concluded we are supposed to have approximately 45 bp because the insert is 15bp + 30 bp on either side of the insert (Xba1 and Spe1 restrictions sites + sticky ends + extra bases). We used the same logic for Lanes 5 and 6 which are supposed to contain approximately 86 base pairs.

One discrepancy in this gel, however, is that Lanes 5 and 6, which according to their labels respectively contain the LacIQ promoter and the Lac IQ1 promoter. According to the information online at the parts registry website, pLacIQ1 should be the same length as pLacIQ. The gels do not show this, but instead show that the sample in Lane 5 is smaller than that in Lane 6 because Lane 5 traveled further. Leland Taylor and Romina Clemente sent out an email to Pallavi Penumetcha to ask about this because she had written a paper about this.

We then unloaded the 0.8% agarose gel and then placed this in the BioRad machine under the same protocol as above. The following image is the 0.8% Agarose.

[[Image:2009-06-09_15hr_21min.jpg]]

We observed that the bands in Lane 1 (which is now at the top) are examples of high intensity bands. The plasmids contained in the original well are twice the intensity of the insert.

In Lane 5, however, we observed several different bands. This could be due to the extra DNA that was sitting on the top of the well. We decided to re-expose the gel to get better observations on Lanes 3 and 4 because the bands were not as intense. We anticipated seeing a band at about 250 bp because this was the size of the insert (plus extra bp). After re-exposing the gel, we did not find any difference and still did not see any bands in these lanes. We have decided to redo the gel on the part S03511 and so we prepared the master mix with that DNA and incubated it so that we could run the gel tomorrow.

We also cut out the "empty" plasmids from the gel so that we could use this as vectors for the coding sequence of the suppressor tRNAs.

Tomorrow we will be planning out specific tasks for people once the oligos arrive.

==Wednesday, June 10, 2009==

We began this morning by making the agarose gel with the same protocol as yesterday. This time, since we are just re-doing both S03511 inserts, we will have a 1.8% agarose gel.

'''OUTLINE: WHERE DO WE STAND?'''

Yesterday
a. Isolated empty plasmids
b. Verified most inserts
c. Waiting for:
>tRNA oligos
>Primers
a. Will amplify the reporter gene in the plasmid itself

Next?
a. When Primers Arrive
> Amplify the reporter genes with primers (PCR takes ~3 hours)
> Calculate how much reporter plasmid we will need (Start with 50x more than we actually will need)
> Cut amplified sequence (EcoR1 and BamH1/Nco1) then purify what we need
> Cut reporter plasmid (EcoR1 and BamH1/Nco1) then purify what we need
> Ligate amplified sequence and reporter plasmid
b. When tRNA oligos arrive
> Calculate how much of each oligo we will need (takes a while...)
> "Boil and cool" tRNA oligos together
> Ligate fragments (dsDNA) into plasmids we isolated yesterday (~5 minutes)
> Transform the plasmids (~20 minutes)

* LOOK THROUGH LIGATION PROTOCOL (How to assemble genes)
* LOOK THROUGH TRANSFORMATION PROTOCOL

''What we need to get done today'':
1. Prepare the control for the Tet Resistance by BioBricking the part pLac+RBS (S03511) with the TetA (J31007).
2. Check the part number for RFP and also for pLac+RBS+RFP
3. Read through ligation protocol
4. See how many nanograms of plasmid we will need for the tRNAs and for the inserts and multiply this number by 50.
> Check if we have enough plasmids from our mini-prep
5. Purify the plasmids from the gel

After we made this outline, Leland, Olivia and I analyzed the gel we ran this morning and did not see a more than one band in the 2 lanes that contained DNA from S03511 (image below). Since we did not see multiple bands in yesterday's gel either, we conclude that there is something wrong with the DNA rather than with the restriction enzymes that we used to cut it. We concluded that there is probably not an insert in this plasmid both the part and the plasmid are useless.

[[Image: 2009-06-10 10hr 30min.jpg]]

Now, we do not have an immediately accessible promoter. We have decided to order the pBAD promoter from Missouri Western and we will attach the RBS+Tet to this plasmid. Our insert for this will be the larger gene because it is easier to manipulate in the gel than the small gene.

Alyndria Thompson and Romina Clemente are figuring out the size of the plasmids including the reporter genes.

After lunch we performed several steps to isolate and purify the gel to get the empty plasmids will be the vectors for the tRNA DNA. Then, we used the NanoDrop to assess the purity of our DNA. We found that the plasmids were more pure than we had expected. The following values were obtained:

Abs 1.850
A-260 10 mm path 0.172
A-280 10 mm path 0.075
260/280 2.28
260/230 0.09

We then followed the protocol for preparing the agar plates. We put the plate with the agar solution in the Autoclave.

While the solution was in the autoclave, we discussed that we don't really need a promoter in front of RBS+Tet for the gene to be expressed because Tet expression is so strong that the gene would translate without a promoter. However, it would be a good idea to have the promoter because if it was possible to reinforce the translation of the reporter protein, we wanted to do that.

We found that there were some tubes in the lab fridge that contained the pBAD and pLac promoters with RBS. We decided to use these as the promoter and RBS for our experiments. The pLac promoter will be used for RFP and the pBad promoter used with Tet Resistance. We decided to use the pBad with Tet because it is weaker in comparison to pLac. As expression of the Tet gene can be toxic to a cell, we do not want to overexpress the gene with a stronger promoter.

Once the agar solution was sterilized in the Autoclave, we removed it, cooled it, and pipetted the antibiotic resistance into it. We then poured the solution into the plates, removed any bubbles, and let the gel form.

Alyndria Thompson and Romina Clemente concluded that we had more than enough recieving vectors for both the RFP and Tetracycline genes. Since we need 50 nanograms, it is wise to have at least 50 times that much that we can work with, so we need at least 2,500 ng.

RFP 1: 77.6 x 4 = 310.4 ng/uL
x 20 uL = 3,272 ng
RFP 2: 59.5 x 4 = 238 ng/uL
x 20 uL = 4,760 ng
Tet: 40.9 x 4 = 163.6 ng/uL
x 20 uL = 3,272 ng

According to these calculations we have more than 50 times what we need for both instances of RFP and Tet.

Tomorrow, we can prepare the Master Mix for the digestions of both reporter plamids and then add the restriction enzymes once they arrive. Then, we can follow through with the rest of the plans we had made above in the section titles "Next?".

==Thursday, June 11, 2009==

We began this morning by calculating how many uL we would need for each of the mixes for digesting our reporter plasmids. We decided to double the final volume of each mix to 40uL instead of 20uL. Since we knew the concentration of each plasmid and also knew we needed 2500 ng, we cross multiplied to find the uL of DNA we would need for each.

Concentration Calculations:
310.4 ng/1 uL = 2500ng/x uL
x = 8.05 uL

Here is a chart indicating how much of each we used for the 2 mixes.

[[Image: DoubledigestRFPTetR.png]]

On the Promega website, we found a list of compatible buffers for double digestions. Buffers E and H are compatible for the Tet plasmid and RFP plasmid digestions respectively. We fortunately have both buffers in stock.

[[Image:BamEcoR1.png]] [[Image:NcoEcoR1.png]]

We waited for our waited for the restriction enzymes to arrive and in the meantime, we transformed the plasmids with pLac+RBS, pBad, and pBad+RBS into ''E.coli'' cells. We want the ''E.coli'' cells to replicate the plasmids with these genes because we have a limited amount of genes. [http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_Transformation.html Zippy Plasmid Transformation]

Once our oligos and restriction enzymes arrived, we got to work right away!

'''PCR (Polymerase Chain Reaction)'''

We calculated how much plasmid we will need for the PCR. We decided to dilute the concentration of our plasmids a hundred fold so that we could get pipetable amounts.

''Change in Concentrations''
310.4 ng/uL x 1/100 (dilution factor) = 3.104 ng/uL (RFP)
163.6 ng/uL x 1/100 (dilution factor) = 0.164 ng/uL (Tet)

''Example shown with RFP''
3.104 ng/1 uL = 1ng/ x uL, x = .32
For Tet, x = .61

We also calculated how much of each primer we would need with the following equation:

C1V1 = C2V2
(100 uM) (x) = (1 uM)( 100 uL)
x = 1 uL

[[Image: screen-capture-8.png]]

Each of the 5 of us made had 2 tubes (one for each reporter protein) because there were 5 different forward primers.To each tube, we added the template DNA (either RFP or Tet), water, the respective reverse primer (either RFP or Tet), and the forward primer depending on which 5mer we had chosen. My 5mer was CCAUC. Finally, we added the Master Mix last because this contained the enzyme (DNA polymerase) that would amplify our desired sequence.

[[Image: Pcrtubes.png]]

We followed the temperature cycles so that PCR could occur. The annealing temperature needed to be 5C less than the smallest melting temperature of the primers. Since the reverse primer of RFP had a melting temp of 66C, we set the temperature of the 3rd cycle on 61C.

[[Image: Temperaturecycle.png]]

'''tRNA Oligo Assembly'''

We needed to calculate the concentration of each of the oligo before carrying out the assembly. The stock concentration of each of the oligo was 100 uM. Since we wanted to have 5uM of the oligo in the test tube, and we know that final volume of the test tube is 20 uL, we just cross multiplied to find the amount we needed to add to the tubes to get this concentration.

(100uM)x = (5uM)(20uL)
x = 1 uL

We added 1 uL of each oligo to the tubes.

We had six total tRNAs we needed to assemble so each of us took one FML (one person took two) and to each tube we added 1uL of each oligo for a total of 7uL, 2uL of buffer, and 11uL of water for a total volume of 20 uL. Five of the oligos were constant and 2 varied for each of the different tRNAs. So everyone pipetted the same 5 oligos and everyone also had 2 unique oligos to add. When all of the oligos were added and the tubes ready, we boiled the tubes at 50C for 5 minutes. We then allowed the tubes to remain in the water to cool very slowly overnight so that the annealing process could take place.

The last thing we did at the end of the day was finish off our plasmid digestion that we had begun in the morning. We added the buffer, DNA, water, and restriction enzymes and placed this in the incubator for overnight digestion.

'''To Do''':

1. Isolate our desired dsDNA from the PCR.
2. Verify our tRNA inserts and eventually transform these into plasmids.
3. Check our agar plates to see colonies that have replicated our pLac+RBS, pBAD+RBS, and pBAD genes.

==Friday, June 12, 2009==

This morning, we began by outlining the main items on today's agenda.

1. tRNAs: Ligation, Transformation
2. Digestion reporters (run all of it and gel purify)
3. Transformed cells
4. PCR: Gel run an aliquot to verify (5uL)

We used the equation given by the Ligation protocol to calculate how many nanograms of insert we would need. Initially, we started out calculated the MW average of the 9 and 10 anticodon loops of tRNAs which were 15828.23 ug/umols and 159016.6 ug/umols respectively. We then saw, however, that these would not be helpful values in the process. Instead we started out with the equation below to find the final ng of insert we would need for the ligation.

[[Image: ligationcalculations.png]]

We calculated our initial concentration in ng/uL of insert as shown below.

[[Image: ligationequation.png]]
X nanograms of insert = [(2)(144bp)(50 ng linearalized plasmid)]/2038bp
X = 7.0657 ng of insert

We wanted to find the amount of uL of insert we would need to use. We diluted the initial concentration 50x because we wanted a pipettable amount of insert. Since we knew we wanted the final amount of nanograms to be 7.0657, we calculated as shown below:

''DILUTION'': 555.48/50 = 11.012
''CALCULATION'': 11.012 ng/ 1uL = 7.065 ng/ x uL
x = .64 uL of insert

After we had done these calculations, half of the group started to carryout the ligation of the tRNA inserts into empty plasmids. We needed 7 ligations total: 6 were the tRNAs we needed, while the last one is a negative control. These are the final amounts of what we used:

[[Image: Trnaligation.png]]

The other half of the group (which included me) prepared the agarose gels so that we could run gels for the digested reporters and the PCR inserts. We calculated that the digested reporters needed an agarose of 4% and the PCR reporters needed an agarose of 1.45%. We allowed this gel to form for about 45 minutes to an hour. We prepared both the gels and allowed them to run through lunch.
After lunch, we took pictures of our gels and noticed that the third lane in the PCR gel (third lane from the top in the bottom photo) did not develop a band. The lane was supposed to contain the CUAGU insert for Tetracycline Resistance.

[[Image: 2009-06-12 15hr 54min.jpg]] [[Image:2009-06-12_16hr_07min.jpg]]

Romina Clemente and I decided to repeat this PCR using the TetResistance and RBS+TetResistance plasmids as templates. We had to recalculate how much template DNA to use. We let the PCR run overnight. We also verified that the digestion fragments (both the plasmid and the insert) on the other gel were the correct size. The plasmids, which are the brighter fragment in the gel with only 2 lanes, contain the plasmid base pairs along with the rest of our reporter genes. We isolated these plasmids from the gel today and stored them for purification on Monday.

In the meantime, other members of our lab had finished the ligation of the tRNA genes to the empty plasmids and were now transforming these into competent cells. They placed the agar plate with these cells in the fridge overnight.

We also observed the transformations that we had performed yesterday and noticed that I13453 did not form any colonies. J04500 and J3102 did form colonies.

To Do:
1. PCR: Dig, Ligate, Transform
Clean and concentration column, digest, clean and concentration column, ligate.
2. Gel purification of our digestion plasmids
3. Check if colonies have grown for our tRNAs

==Saturday, June 13, 2009==

Alyndria Thompson came in on Saturday morning to remove the tRNA plates and put the PCR reactions in the fridge.

'''tRNA Colonies'''

She counted the colonies of tRNA on each plate.

[[Image:TRNA_colonies_6_13_09.png]]

She placed the plates in the large fridge on the top shelf at 9:23 am. She talked to Dr. Campbell who said we will have to try to "work them up".

'''PCR'''

Products were placed in orange rack of smaller fridge. [2*, 2!]

File:TRNA colonies 6 13 09.png

2009-06-15T00:39:16Z

ShPunjabi:

IGEM Notebook

2009-06-15T00:38:19Z

ShPunjabi: /* Friday, June 12, 2009 */

==Wednesday, June 3, 2009==

Romina Clemente and I are trying to find suitable reporter proteins to use. Yesterday, Leland Taylor and Alyndria Thompson were working on ways to insert the gene sequences into the plasmid. Upon seeing how they wanted to manipulated the reporter gene to include the logical clauses, we came up with a few criteria for the reporter genes we would use. The following criteria for genes are listed in order of the broadest aspect to look at to the narrowest aspect:
a) Doesn't contain restriction sites for the 4 restriction enzymes (EcoR1, Xbal, Spel, Pst1) used to cleave the Biobrick part out of the plasmid.
b) Contains 6 cutter restriction sites.
c) These restriction enzymes aren't blunt (cleave straight down at one spot).
d) These restriction sites are close to thge 5' (beginning) end of the sequence.
e) These enzymes are easiest to work with and cheapest.
We are finding the part numbers of the reporter genes we want to use (antibiotic resistance, fluorescence, LacZ) through our own GCAT because we know these ones work. We are then locating these parts on the parts registry [1] website. We copied and pasted the gene sequences we obtained from the registry onto the ApE software [2]. From here, we were able to generate a genetic map of each gene that outlined each restriction site that fit our criteria. We put each genetic map, alongside the part number used, into a Word document.

http://gcat.davidson.edu/GcatWiki/images/0/0e/Restriction_Site_Mapping_on_Reporter_Genes.doc

Later on in the day, we decided that the best way to test for suppression would involve placing the 5mers at the beginning of the reporter gene instead of inserting them into the reporter gene. To do this, we decided that the gene along with its start codon need to be expressed after the 5mer. We will insert a BioBrick plasmid with a 5mer and start codon that will be incorporated before the gene:
RBS-6-8nt-ATG-5mer--ATG-gene

Leland Taylor assembled the oligos that we will need for the BioBrick pieces.

In order to test whether the reporter protein would be expressed regardless of the 5mer being suppressed or not, we would need to remove the suppressor tRNAs from the cell.

Meanwhile, I helped figure out the coding sequence that we will need for the suppressor tRNAs that will bind to the 5mers. We decided that we would be using the following suppressor tRNAs on the Davidson side: CUAGU, CCCUC, CGGUC, CCAUC, and CCACU. We searched through several papers, with little result and finally emailed Dr. Christopher Anderson of UC Berkeley to request the DNA sequence. He emailed us back with a generic DNA code that we could use to create the tRNA; however, for each suppressor tRNA we would use, we would need to change base pairs in the anticodon loop.

The length of the tRNA is 92 nt. The anticodon loop is comprised of 9 nt. Once we have BioBricked the DNA sequence with 4 restriction sites and supplemental nucleotides, the length of the entire gene is 144 nucleotides. This is made up of the 92 nt +22 nt on each side of the sequence (44) + 4 nt on each side of the restriction ends (8).

At the end of the day, we decided to use tetracycline resistance and RFP as our reporter proteins for this tester experiment.

==Thursday, June 4, 2009==

This morning, I began the day by learning how to use the PEARL prgram. Olivia Ho-Shing sat with me and walked me through the program script. And then, Leland Taylor and Shashank Suresh had a problem they wanted us to solve. Leland explained that we need to make sure there is a stop codon in the gene if we fail to suppress the 5mer. The stop codon needs to be before we find another 5mer. They also wanted us to run through both the RFP and Tet Resistance genes to make sure that they did not contain any of the anticodon sequences from our 5 frameshift suppressor tRNAs so as to throw the translation out of frame. They further wanted us to confirm that if we do have successful suppression, there are no stop codons in the middle of the RFP and Tet Resistance genes. PEARL did not find any matches for the suppressor tRNAs within genes. We found 3 matches for stop codons UAA. Two of these occured at the end of the Tet and one at the end of RFP, where they should normally be to end translation into a protein.

Following this, Olivia Ho-Shing and I tried to determine how we can add BioBrick ends to our single stranded DNA sequences that code for the suppressor tRNAs. Our ultimate goal is to find complements for these single strands so they can be put into plasmids. We found the standard prefixes and suffixes for sequences that do not contain ATG (becuase this sequence is a functional RNA and will not be translated) and we placed these before and after the altered 92bp sequence that Dr. Anderson provided us with. The prefixes and suffixes are necessary because they are the extra nucleotides that will be mimicking a "restriction enzyme cutting site". The single strand appeared as below after the Bio Brick prefixes and suffixes were added:

22ntPREFIX--32nt--VARIABLE ANTICODON LOOP--51nt--21ntSUFFIX

We used the suppressor codon CUAGU as our example and placed its appropriate anticodon loop in the sequence. We put this into the lancelator that would give us the other strand as well as the oligos we would need for ideal construction of this double stranded DNA fragment to put into the plasmid. We recieved a total of 4 oligos, 2 of which were variable and 2 of which were constant for all 6 different suppressor tRNAs. Using this double strand, we simulated a restriction enzyme digestion by EcoRI and PstI. We took off the G at both ends of the top strand and then removed the first five nucleotides on each end of the bottom strand. This made the top strand longer than the bottom one. We then proceeded to take the two variable strands and changed the anticodon loop nt for each suppressor tRNA we used.

Around lunchtime, we encountered a problem with the BioBrick scar in the "ATG-5mer subpiece" of the test project. It turns out that after annealing the sticky ends of the Xba1 and Spe1 sites, the scar created had a stop codon TAG (UAG) that was in frame with the suppressed codon. When the Xba1 site on insert anneals to the Spe1 site on the plasmid (the green portions), we get a scar that reads TACTAG.
[[Image:screen-capture.png]]

For the afternoon, Olivia Ho-Shing and I finished assembly of the oligos of the suppressor tRNA codes. We assembled the document below:

http://gcat.davidson.edu/GcatWiki/images/5/5e/TRNAoligostoOrder.doc

We were assigned tasks of creating "cartoons" to illustrate the phenomenon that had occurred. No one could successfully do this however, because we could not understand how the piece had formed. The parts registry website provided wrong information on the restriction site of Spe1 which slowed our efficiency. This project turned into an effort to try to find two other different restriction enzymes that have complementary sticky ends other than Xba1 and Spe1. We want to get rid oc the scar that contains TACTAG.

We ended up deciding that a hybrid of two ideas was needed: restriction enzymes would need to be used to make several reporter gene sequences with different beginnings dependending on the suppressor tRNA sequence needed.

==Friday, June 5, 2009==

Our goal for this morning was to discuss the hybrid PCR approach to assemble the part that comes before the gene we wanted to express. We decided that we would have a BioBrick prefix and a 5mer followed by a sequence of nucleotides whose identity was up to us. Using PCR we would get the second double strand if we have the following:

BioBrick Prefix--ATG--5mer--gene until we get to restriction site

The PCR primer would attach to the ATG and the 5mer and would replicate the complementary base pairs to make a double stranded DNA sequence. As several of us were confused about PCR methods, we looked at the online link provided by Alyndria Thompson's link to the PCR interactive website which was very helpful. We also did some research to discover that the restriction enzyme BseRI (which is contained in the YFP that we wanted to alter) is not compatible with any other restriction enzyme. This is good because we did not want to plasmid containing this gene to close up with Xba1 once we digested it.

The team experienced some confusion with the hybrid idea and spoke to Clif Davis of Missouri Western University on the phone. He told us that there will be one part that is a fragment of the beginning of some nucleotides as a restriction cleaving site, ATG, the logical clause, and the start of the YFP gene that will be placed into a plasmid containing a space. To the left of the space will be the BioBrick Prefix and the right of the space will contain the rest of the genes in the YFP. Therefore, we decided not to alter any BioBrick ends and just look at restriction sites at the beginning of reporter proteins. The small fragment that will be placed into the plasmid will be made by PCR. The PCR will attach a primer onto the ATG and 5mer and replicate.

While we were discussing this, we expanded upon the idea that we can do whatever we want with the "suffix" portion between the 5mer and the beginning of YFP. We decided it would be best to remove ATG and add the first 5 amino acids and the restriction enzyme site. Along with this, if we have the 5mer coding for a certain amino acid (ie methionine) we could replace another codon that codes for methionine WITH that 5mer so long as this codon is contained within the first 15 nt. This way, we would not be altering the protein.

Olivia Ho-Shing came up with an idea of cascading PCR effect for longer logical clauses. Post translational modification of the protein was another one of her ideas. This applies because although we may code for the protein in a given way, the protein may modify or fix itself up to perform a slightly different function in its tertiary form.

Dr. Campbell suggested that we can look up restriction enzymes 400-500 base pairs into the reporter protein we would like to use; however, we have more flexibility in our procedure if we choose more "hardy" cutters. So instead, we could cut with EcoRI instead of with Xba1.

During the afternoon, we set about three tasks:
1) Which primers to use
2) Which promoters to use
3) Which restriction enzymes and how they will determine use of reporter proteins

Olivia Ho-Shing and I began looking up which restriction enzymes were strong so we could base our reporters on that. We began with Tet and RFP since those were the two reporters we had started out with choosing. I found a website that provided enzyme cleavage efficiency based upon the proximity of the site to the end of the PCR fragment that contains it. We discovered the following based upon the attached documents:

http://gcat.davidson.edu/GcatWiki/images/8/8f/Restriction_Enzyme_Cleavage_Efficiency.doc
http://gcat.davidson.edu/GcatWiki/images/9/9a/07Jun09_RE_Cutting_Efficiency_oh.doc

We decided that these two reporters contained efficient enzymes in a good quantity to choose from. On Monday, we will look through these enzymes once more and finalize which enzymes we would like to use with RFP and Tet.

==Sunday, June 7, 2009==

We did preparations for the mini prep this evening. The following notes are courtesy of Olivia Ho-Shing.

1. Sterically transfer ~2mL LB+AMP (AMP are ampicillin resistant plasmids) *Never put fluid back into bottle once it has entered pipette*
2. Use forceps to grab toothpick, scrap some frozen cells, rub in tube with top

'''Tubes'''

S03511 (2)

K091111

B0030

I715039

K091112

I715039

J31007 (2)

B0034

E1010

S03710

==Monday, June 8, 2009==

We reviewed the PCR process this morning as well as the restriction enzyme cleavage process.

Then, Leland Taylor, Olivia Ho-Shing and I all worked on converting the tRNA oligos we made to the template that Missouri Western used. The oligos we had previously made were different from the ones that Dr. Anderson had originally emailed us because further communication between Dr. Anderson and Missouri Western resulted in a better template.

We tried several times to minimize the number of oligos while keeping their individual lengths under 80bp. We managed to do this in the end by creating 7 oligos, 2 of which are variable between the suppressor tRNAs.
We reviewed the following document and finalized the oligos that we needed to order for the frameshift suppressor tRNAs. We decided to keep both the 10bp and 9bp anticodons of CCAUC.

I also looked up the amino acids that corresponded to the FS (frameshift suppressor) tRNAs for future use:

1. CUAGU = Leu
2. CCCUC = Pro
3. CGGUC = Arg
4. CCACU = Pro
5. CCAUC* = Pro
*9 and 10 bp anticodons

Also, we have decided to use NcoI as the restriction enzyme for RFP. This cleavage site occurs 419 bp into the gene. We have decided to use BamHI as the enzyme for Tetracycline Resistance and the cleavage site occurs 290bp into the gene. Both of these enzymes are very powerful and hardy.

We also looked over Missouri Western's tRNA oligos and primer oligos which we approved.
Our oligos were also approved by Missouri Western and Dr. Campbell ordered them while we supervised.

In the afternoon we did the miniprep. The mini prep directions were as appear in the link below.

http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_MiniPrep.html

We then worked with the Nano Prep to dilute our solution of DNA to an absorption of 1.5 and then we made labels for the tubes. The chart below shows relevant information we obtained from the NanoDrop for each of the reporter plasmids.

[[Image: Screen-capture-6.png]]

==Tuesday, June 9, 2009==

'''Preparations for restriction enzymes digestion of plasmids'''

Purpose: To determine the size of the plasmid inserts and ensure that plasmids are what we extracted from the cells during the mini-prep.

Things we need:
DNA ~3uL
Buffer 10x (Want to dilute to 1x) so add 2uL
Enzyme 1 ~1uL
Enzme 2 ~1uL
Water ~13uL
Final Volunter about 20 uL.

Need to choose the right buffer for the restriction enzymes. All buffers come in 10x (10 times more concentrated than what you want it to be when you use it). [http://gcat.davidson.edu/GcatWiki/index.php/Davidson_Missouri_W/Double_Digest_Guide Buffer Website]

Total volume of enzymes has to be 10% or less than final volume (most volume 2uL). Barely submerge the pipet tip into the solution when pipeting the enzymes.
Star (*) activity: Restriction enzyme stops cutting specifically and starts cutting everywhere.

DNA can't be seen by itself so we add EtBr (the bigger the insert, the more will bind) so bigger strands appear brighter. samples.

The order of entry doesn't matter, but make sure to enter the enzymes last.

Will have lots of labeled tubes and will put 3uL of appropriate plasmids into the tubes.
In the meantime, we'll make a "Master Mix" of all the other ingredients in a bigger tube. Always make enough volume for one extra tube.

Enzymes usually have an optimal temperature of 37C. We will let this reaction go for an hour and then we will run it on a gel.

'''Gel Electrophoresis Protocol'''

Website online for ideal gel concentration for resolving different sizes of molecules [http://gcat.davidson.edu/iGEM08/gelwebsite/gelwebsite.html Gel Concentration Website].
See protocol on the Davidson/Missouri Western Wookie for running molecules in the gel and how to digest with restriction enzymes.

'''What's Next?'''

1. 5' end additions
a. Using PCR
b. Cut at the restriction sites EcoR1 and either Bam H1 or Nco1
c. Cut plasmid and reporter with same restriction enzymes as above
d. Run a gel and purify "keeper" DNA (Ligate transform, screen, sequence)
e. Then, we take the "keeper" DNA and combine it with the insert from part

We can have steps d and e ready in advance.

2. tRNAs
a. Assemble the oligos into genes (Requires lots of calculations beforehand, but the actual steps )
b. Plasmids cut up with EcoR1 and Pst1 '''WE CAN USE THE "WASTE" PLASMID FROM THE RESTRICTION ENZYME DIGEST'''
c. Gel purify the plasmids (Ligate transform, screen, sequence)

The following tables include Master Mix measurements, agarose gel concentrations and buffers.
http://gcat.davidson.edu/GcatWiki/images/0/08/Shamitapic3.png
http://gcat.davidson.edu/GcatWiki/images/d/d1/Shamitapic4.png
http://gcat.davidson.edu/GcatWiki/images/6/6f/Shamitapic5.png

We made the Master Mix with our DNA and put it in the incubator at 10:40 AM.

'''Preparing the Gels'''

Now, we will be making 2 gels, 1 at 0.8% and 2.5% (weight to volume percent). The gel volume is 60mL.

'''Definition of 1% solution:
1 gram of agarose/ 100mL buffer'''

So for a 0.8% solution: 0.8 grams agarose/100mL buffer AND we need 60mL (fixed volume for casts run) so multiply that by 0.6.
Final concentration:

.48 grams agarose/60mL buffer

1 uL of EtBr stock for 60mL of gel.
EtBr is in the buffer so it is constantly replenishing itself even though it is moving toward one end in order to make bands on the other end visible.

After Dr. Campbell's demonstration, we each helped prepare a 0.8% buffer solution using the agarose powder, buffer and EtBr. We mixed together the first two substances in a 200mL Erlenmeyer flask and heated it for 120 seconds in the microwave. We then added EtBr and poured the gel solution into the gel cast.

'''Agar Plate'''

After lunch, we grew some more cells for each of the 11 parts in an agar plate labeled "LBAMP-AMC-9 June" so that so would have extra cells that could be stored for up to 2 weeks.
We made small circles on the agar plate and numbered them 1-11. The numbers corresponded to the following parts. We put the plate in the incubator overnight to let the cells grow.

1. B0030 AMC
2. K091111
3. S03710
4. I715039 OEH
5. S03511 ORH
6. I715039 AST
7. B0030 SDP
8. K091112
9. J31007 LJH
10. J31007 REC
11. S03511

'''Unloading the Gels'''

The following shows which parts we entered in which gels:

[[Image: screen-capture-2.png]]

We unloaded the 2.5% agarose first and after observing the bands under a UV light, put the gel into the BioRad machine to observe the bands. The image below is of a Molecular Weight Marker that shows the number of bp in control fragments of DNA according to the distance they travel down the gel.

[[Image: DNA_molecular_weight_marker_gel.png]]

We obtained the following printout for the first gel that was 2.5% Agarose.
[[Image: 2009-06-09_14hr_44min.jpg]]

The bands in the first well at the bottom are those of the molecular weight marker. We skipped the lane above (to the right) of the MWM and entered the rest of the inserts as described in the above table. A light band is visible for each of the 4 other wells. In Lanes 3 and 4 we concluded we are supposed to have approximately 45 bp because the insert is 15bp + 30 bp on either side of the insert (Xba1 and Spe1 restrictions sites + sticky ends + extra bases). We used the same logic for Lanes 5 and 6 which are supposed to contain approximately 86 base pairs.

One discrepancy in this gel, however, is that Lanes 5 and 6, which according to their labels respectively contain the LacIQ promoter and the Lac IQ1 promoter. According to the information online at the parts registry website, pLacIQ1 should be the same length as pLacIQ. The gels do not show this, but instead show that the sample in Lane 5 is smaller than that in Lane 6 because Lane 5 traveled further. Leland Taylor and Romina Clemente sent out an email to Pallavi Penumetcha to ask about this because she had written a paper about this.

We then unloaded the 0.8% agarose gel and then placed this in the BioRad machine under the same protocol as above. The following image is the 0.8% Agarose.

[[Image:2009-06-09_15hr_21min.jpg]]

We observed that the bands in Lane 1 (which is now at the top) are examples of high intensity bands. The plasmids contained in the original well are twice the intensity of the insert.

In Lane 5, however, we observed several different bands. This could be due to the extra DNA that was sitting on the top of the well. We decided to re-expose the gel to get better observations on Lanes 3 and 4 because the bands were not as intense. We anticipated seeing a band at about 250 bp because this was the size of the insert (plus extra bp). After re-exposing the gel, we did not find any difference and still did not see any bands in these lanes. We have decided to redo the gel on the part S03511 and so we prepared the master mix with that DNA and incubated it so that we could run the gel tomorrow.

We also cut out the "empty" plasmids from the gel so that we could use this as vectors for the coding sequence of the suppressor tRNAs.

Tomorrow we will be planning out specific tasks for people once the oligos arrive.

==Wednesday, June 10, 2009==

We began this morning by making the agarose gel with the same protocol as yesterday. This time, since we are just re-doing both S03511 inserts, we will have a 1.8% agarose gel.

'''OUTLINE: WHERE DO WE STAND?'''

Yesterday
a. Isolated empty plasmids
b. Verified most inserts
c. Waiting for:
>tRNA oligos
>Primers
a. Will amplify the reporter gene in the plasmid itself

Next?
a. When Primers Arrive
> Amplify the reporter genes with primers (PCR takes ~3 hours)
> Calculate how much reporter plasmid we will need (Start with 50x more than we actually will need)
> Cut amplified sequence (EcoR1 and BamH1/Nco1) then purify what we need
> Cut reporter plasmid (EcoR1 and BamH1/Nco1) then purify what we need
> Ligate amplified sequence and reporter plasmid
b. When tRNA oligos arrive
> Calculate how much of each oligo we will need (takes a while...)
> "Boil and cool" tRNA oligos together
> Ligate fragments (dsDNA) into plasmids we isolated yesterday (~5 minutes)
> Transform the plasmids (~20 minutes)

* LOOK THROUGH LIGATION PROTOCOL (How to assemble genes)
* LOOK THROUGH TRANSFORMATION PROTOCOL

''What we need to get done today'':
1. Prepare the control for the Tet Resistance by BioBricking the part pLac+RBS (S03511) with the TetA (J31007).
2. Check the part number for RFP and also for pLac+RBS+RFP
3. Read through ligation protocol
4. See how many nanograms of plasmid we will need for the tRNAs and for the inserts and multiply this number by 50.
> Check if we have enough plasmids from our mini-prep
5. Purify the plasmids from the gel

After we made this outline, Leland, Olivia and I analyzed the gel we ran this morning and did not see a more than one band in the 2 lanes that contained DNA from S03511 (image below). Since we did not see multiple bands in yesterday's gel either, we conclude that there is something wrong with the DNA rather than with the restriction enzymes that we used to cut it. We concluded that there is probably not an insert in this plasmid both the part and the plasmid are useless.

[[Image: 2009-06-10 10hr 30min.jpg]]

Now, we do not have an immediately accessible promoter. We have decided to order the pBAD promoter from Missouri Western and we will attach the RBS+Tet to this plasmid. Our insert for this will be the larger gene because it is easier to manipulate in the gel than the small gene.

Alyndria Thompson and Romina Clemente are figuring out the size of the plasmids including the reporter genes.

After lunch we performed several steps to isolate and purify the gel to get the empty plasmids will be the vectors for the tRNA DNA. Then, we used the NanoDrop to assess the purity of our DNA. We found that the plasmids were more pure than we had expected. The following values were obtained:

Abs 1.850
A-260 10 mm path 0.172
A-280 10 mm path 0.075
260/280 2.28
260/230 0.09

We then followed the protocol for preparing the agar plates. We put the plate with the agar solution in the Autoclave.

While the solution was in the autoclave, we discussed that we don't really need a promoter in front of RBS+Tet for the gene to be expressed because Tet expression is so strong that the gene would translate without a promoter. However, it would be a good idea to have the promoter because if it was possible to reinforce the translation of the reporter protein, we wanted to do that.

We found that there were some tubes in the lab fridge that contained the pBAD and pLac promoters with RBS. We decided to use these as the promoter and RBS for our experiments. The pLac promoter will be used for RFP and the pBad promoter used with Tet Resistance. We decided to use the pBad with Tet because it is weaker in comparison to pLac. As expression of the Tet gene can be toxic to a cell, we do not want to overexpress the gene with a stronger promoter.

Once the agar solution was sterilized in the Autoclave, we removed it, cooled it, and pipetted the antibiotic resistance into it. We then poured the solution into the plates, removed any bubbles, and let the gel form.

Alyndria Thompson and Romina Clemente concluded that we had more than enough recieving vectors for both the RFP and Tetracycline genes. Since we need 50 nanograms, it is wise to have at least 50 times that much that we can work with, so we need at least 2,500 ng.

RFP 1: 77.6 x 4 = 310.4 ng/uL
x 20 uL = 3,272 ng
RFP 2: 59.5 x 4 = 238 ng/uL
x 20 uL = 4,760 ng
Tet: 40.9 x 4 = 163.6 ng/uL
x 20 uL = 3,272 ng

According to these calculations we have more than 50 times what we need for both instances of RFP and Tet.

Tomorrow, we can prepare the Master Mix for the digestions of both reporter plamids and then add the restriction enzymes once they arrive. Then, we can follow through with the rest of the plans we had made above in the section titles "Next?".

==Thursday, June 11, 2009==

We began this morning by calculating how many uL we would need for each of the mixes for digesting our reporter plasmids. We decided to double the final volume of each mix to 40uL instead of 20uL. Since we knew the concentration of each plasmid and also knew we needed 2500 ng, we cross multiplied to find the uL of DNA we would need for each.

Concentration Calculations:
310.4 ng/1 uL = 2500ng/x uL
x = 8.05 uL

Here is a chart indicating how much of each we used for the 2 mixes.

[[Image: DoubledigestRFPTetR.png]]

On the Promega website, we found a list of compatible buffers for double digestions. Buffers E and H are compatible for the Tet plasmid and RFP plasmid digestions respectively. We fortunately have both buffers in stock.

[[Image:BamEcoR1.png]] [[Image:NcoEcoR1.png]]

We waited for our waited for the restriction enzymes to arrive and in the meantime, we transformed the plasmids with pLac+RBS, pBad, and pBad+RBS into ''E.coli'' cells. We want the ''E.coli'' cells to replicate the plasmids with these genes because we have a limited amount of genes. [http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_Transformation.html Zippy Plasmid Transformation]

Once our oligos and restriction enzymes arrived, we got to work right away!

'''PCR (Polymerase Chain Reaction)'''

We calculated how much plasmid we will need for the PCR. We decided to dilute the concentration of our plasmids a hundred fold so that we could get pipetable amounts.

''Change in Concentrations''
310.4 ng/uL x 1/100 (dilution factor) = 3.104 ng/uL (RFP)
163.6 ng/uL x 1/100 (dilution factor) = 0.164 ng/uL (Tet)

''Example shown with RFP''
3.104 ng/1 uL = 1ng/ x uL, x = .32
For Tet, x = .61

We also calculated how much of each primer we would need with the following equation:

C1V1 = C2V2
(100 uM) (x) = (1 uM)( 100 uL)
x = 1 uL

[[Image: screen-capture-8.png]]

Each of the 5 of us made had 2 tubes (one for each reporter protein) because there were 5 different forward primers.To each tube, we added the template DNA (either RFP or Tet), water, the respective reverse primer (either RFP or Tet), and the forward primer depending on which 5mer we had chosen. My 5mer was CCAUC. Finally, we added the Master Mix last because this contained the enzyme (DNA polymerase) that would amplify our desired sequence.

[[Image: Pcrtubes.png]]

We followed the temperature cycles so that PCR could occur. The annealing temperature needed to be 5C less than the smallest melting temperature of the primers. Since the reverse primer of RFP had a melting temp of 66C, we set the temperature of the 3rd cycle on 61C.

[[Image: Temperaturecycle.png]]

'''tRNA Oligo Assembly'''

We needed to calculate the concentration of each of the oligo before carrying out the assembly. The stock concentration of each of the oligo was 100 uM. Since we wanted to have 5uM of the oligo in the test tube, and we know that final volume of the test tube is 20 uL, we just cross multiplied to find the amount we needed to add to the tubes to get this concentration.

(100uM)x = (5uM)(20uL)
x = 1 uL

We added 1 uL of each oligo to the tubes.

We had six total tRNAs we needed to assemble so each of us took one FML (one person took two) and to each tube we added 1uL of each oligo for a total of 7uL, 2uL of buffer, and 11uL of water for a total volume of 20 uL. Five of the oligos were constant and 2 varied for each of the different tRNAs. So everyone pipetted the same 5 oligos and everyone also had 2 unique oligos to add. When all of the oligos were added and the tubes ready, we boiled the tubes at 50C for 5 minutes. We then allowed the tubes to remain in the water to cool very slowly overnight so that the annealing process could take place.

The last thing we did at the end of the day was finish off our plasmid digestion that we had begun in the morning. We added the buffer, DNA, water, and restriction enzymes and placed this in the incubator for overnight digestion.

'''To Do''':

1. Isolate our desired dsDNA from the PCR.
2. Verify our tRNA inserts and eventually transform these into plasmids.
3. Check our agar plates to see colonies that have replicated our pLac+RBS, pBAD+RBS, and pBAD genes.

==Friday, June 12, 2009==

This morning, we began by outlining the main items on today's agenda.

1. tRNAs: Ligation, Transformation
2. Digestion reporters (run all of it and gel purify)
3. Transformed cells
4. PCR: Gel run an aliquot to verify (5uL)

We used the equation given by the Ligation protocol to calculate how many nanograms of insert we would need. Initially, we started out calculated the MW average of the 9 and 10 anticodon loops of tRNAs which were 15828.23 ug/umols and 159016.6 ug/umols respectively. We then saw, however, that these would not be helpful values in the process. Instead we started out with the equation below to find the final ng of insert we would need for the ligation.

[[Image: ligationcalculations.png]]

We calculated our initial concentration in ng/uL of insert as shown below.

[[Image: ligationequation.png]]
X nanograms of insert = [(2)(144bp)(50 ng linearalized plasmid)]/2038bp
X = 7.0657 ng of insert

We wanted to find the amount of uL of insert we would need to use. We diluted the initial concentration 50x because we wanted a pipettable amount of insert. Since we knew we wanted the final amount of nanograms to be 7.0657, we calculated as shown below:

''DILUTION'': 555.48/50 = 11.012
''CALCULATION'': 11.012 ng/ 1uL = 7.065 ng/ x uL
x = .64 uL of insert

After we had done these calculations, half of the group started to carryout the ligation of the tRNA inserts into empty plasmids. We needed 7 ligations total: 6 were the tRNAs we needed, while the last one is a negative control. These are the final amounts of what we used:

[[Image: Trnaligation.png]]

The other half of the group (which included me) prepared the agarose gels so that we could run gels for the digested reporters and the PCR inserts. We calculated that the digested reporters needed an agarose of 4% and the PCR reporters needed an agarose of 1.45%. We allowed this gel to form for about 45 minutes to an hour. We prepared both the gels and allowed them to run through lunch.
After lunch, we took pictures of our gels and noticed that the third lane in the PCR gel (third lane from the top in the bottom photo) did not develop a band. The lane was supposed to contain the CUAGU insert for Tetracycline Resistance.

[[Image: 2009-06-12 15hr 54min.jpg]] [[Image:2009-06-12_16hr_07min.jpg]]

Romina Clemente and I decided to repeat this PCR using the TetResistance and RBS+TetResistance plasmids as templates. We had to recalculate how much template DNA to use. We let the PCR run overnight. We also verified that the digestion fragments (both the plasmid and the insert) on the other gel were the correct size. The plasmids, which are the brighter fragment in the gel with only 2 lanes, contain the plasmid base pairs along with the rest of our reporter genes. We isolated these plasmids from the gel today and stored them for purification on Monday.

In the meantime, other members of our lab had finished the ligation of the tRNA genes to the empty plasmids and were now transforming these into competent cells. They placed the agar plate with these cells in the fridge overnight.

We also observed the transformations that we had performed yesterday and noticed that I13453 did not form any colonies. J04500 and J3102 did form colonies.

To Do:
1. PCR: Dig, Ligate, Transform
Clean and concentration column, digest, clean and concentration column, ligate.
2. Gel purification of our digestion plasmids
3. Check if colonies have grown for our tRNAs

==Saturday, June 13, 2009==

Alyndria Thompson came in on Saturday morning to remove the tRNA plates and put the PCR reactions in the fridge.

She counted the colonies of tRNA on each plate.

She placed the plates in the large fridge on the top shelf at 9:23 am. She talked to Dr. Campbell who said we will have to try to "work them up".

IGEM Notebook

2009-06-14T22:04:38Z

ShPunjabi: /* Tuesday, June 9, 2009 */

==Wednesday, June 3, 2009==

Romina Clemente and I are trying to find suitable reporter proteins to use. Yesterday, Leland Taylor and Alyndria Thompson were working on ways to insert the gene sequences into the plasmid. Upon seeing how they wanted to manipulated the reporter gene to include the logical clauses, we came up with a few criteria for the reporter genes we would use. The following criteria for genes are listed in order of the broadest aspect to look at to the narrowest aspect:
a) Doesn't contain restriction sites for the 4 restriction enzymes (EcoR1, Xbal, Spel, Pst1) used to cleave the Biobrick part out of the plasmid.
b) Contains 6 cutter restriction sites.
c) These restriction enzymes aren't blunt (cleave straight down at one spot).
d) These restriction sites are close to thge 5' (beginning) end of the sequence.
e) These enzymes are easiest to work with and cheapest.
We are finding the part numbers of the reporter genes we want to use (antibiotic resistance, fluorescence, LacZ) through our own GCAT because we know these ones work. We are then locating these parts on the parts registry [1] website. We copied and pasted the gene sequences we obtained from the registry onto the ApE software [2]. From here, we were able to generate a genetic map of each gene that outlined each restriction site that fit our criteria. We put each genetic map, alongside the part number used, into a Word document.

http://gcat.davidson.edu/GcatWiki/images/0/0e/Restriction_Site_Mapping_on_Reporter_Genes.doc

Later on in the day, we decided that the best way to test for suppression would involve placing the 5mers at the beginning of the reporter gene instead of inserting them into the reporter gene. To do this, we decided that the gene along with its start codon need to be expressed after the 5mer. We will insert a BioBrick plasmid with a 5mer and start codon that will be incorporated before the gene:
RBS-6-8nt-ATG-5mer--ATG-gene

Leland Taylor assembled the oligos that we will need for the BioBrick pieces.

In order to test whether the reporter protein would be expressed regardless of the 5mer being suppressed or not, we would need to remove the suppressor tRNAs from the cell.

Meanwhile, I helped figure out the coding sequence that we will need for the suppressor tRNAs that will bind to the 5mers. We decided that we would be using the following suppressor tRNAs on the Davidson side: CUAGU, CCCUC, CGGUC, CCAUC, and CCACU. We searched through several papers, with little result and finally emailed Dr. Christopher Anderson of UC Berkeley to request the DNA sequence. He emailed us back with a generic DNA code that we could use to create the tRNA; however, for each suppressor tRNA we would use, we would need to change base pairs in the anticodon loop.

The length of the tRNA is 92 nt. The anticodon loop is comprised of 9 nt. Once we have BioBricked the DNA sequence with 4 restriction sites and supplemental nucleotides, the length of the entire gene is 144 nucleotides. This is made up of the 92 nt +22 nt on each side of the sequence (44) + 4 nt on each side of the restriction ends (8).

At the end of the day, we decided to use tetracycline resistance and RFP as our reporter proteins for this tester experiment.

==Thursday, June 4, 2009==

This morning, I began the day by learning how to use the PEARL prgram. Olivia Ho-Shing sat with me and walked me through the program script. And then, Leland Taylor and Shashank Suresh had a problem they wanted us to solve. Leland explained that we need to make sure there is a stop codon in the gene if we fail to suppress the 5mer. The stop codon needs to be before we find another 5mer. They also wanted us to run through both the RFP and Tet Resistance genes to make sure that they did not contain any of the anticodon sequences from our 5 frameshift suppressor tRNAs so as to throw the translation out of frame. They further wanted us to confirm that if we do have successful suppression, there are no stop codons in the middle of the RFP and Tet Resistance genes. PEARL did not find any matches for the suppressor tRNAs within genes. We found 3 matches for stop codons UAA. Two of these occured at the end of the Tet and one at the end of RFP, where they should normally be to end translation into a protein.

Following this, Olivia Ho-Shing and I tried to determine how we can add BioBrick ends to our single stranded DNA sequences that code for the suppressor tRNAs. Our ultimate goal is to find complements for these single strands so they can be put into plasmids. We found the standard prefixes and suffixes for sequences that do not contain ATG (becuase this sequence is a functional RNA and will not be translated) and we placed these before and after the altered 92bp sequence that Dr. Anderson provided us with. The prefixes and suffixes are necessary because they are the extra nucleotides that will be mimicking a "restriction enzyme cutting site". The single strand appeared as below after the Bio Brick prefixes and suffixes were added:

22ntPREFIX--32nt--VARIABLE ANTICODON LOOP--51nt--21ntSUFFIX

We used the suppressor codon CUAGU as our example and placed its appropriate anticodon loop in the sequence. We put this into the lancelator that would give us the other strand as well as the oligos we would need for ideal construction of this double stranded DNA fragment to put into the plasmid. We recieved a total of 4 oligos, 2 of which were variable and 2 of which were constant for all 6 different suppressor tRNAs. Using this double strand, we simulated a restriction enzyme digestion by EcoRI and PstI. We took off the G at both ends of the top strand and then removed the first five nucleotides on each end of the bottom strand. This made the top strand longer than the bottom one. We then proceeded to take the two variable strands and changed the anticodon loop nt for each suppressor tRNA we used.

Around lunchtime, we encountered a problem with the BioBrick scar in the "ATG-5mer subpiece" of the test project. It turns out that after annealing the sticky ends of the Xba1 and Spe1 sites, the scar created had a stop codon TAG (UAG) that was in frame with the suppressed codon. When the Xba1 site on insert anneals to the Spe1 site on the plasmid (the green portions), we get a scar that reads TACTAG.
[[Image:screen-capture.png]]

For the afternoon, Olivia Ho-Shing and I finished assembly of the oligos of the suppressor tRNA codes. We assembled the document below:

http://gcat.davidson.edu/GcatWiki/images/5/5e/TRNAoligostoOrder.doc

We were assigned tasks of creating "cartoons" to illustrate the phenomenon that had occurred. No one could successfully do this however, because we could not understand how the piece had formed. The parts registry website provided wrong information on the restriction site of Spe1 which slowed our efficiency. This project turned into an effort to try to find two other different restriction enzymes that have complementary sticky ends other than Xba1 and Spe1. We want to get rid oc the scar that contains TACTAG.

We ended up deciding that a hybrid of two ideas was needed: restriction enzymes would need to be used to make several reporter gene sequences with different beginnings dependending on the suppressor tRNA sequence needed.

==Friday, June 5, 2009==

Our goal for this morning was to discuss the hybrid PCR approach to assemble the part that comes before the gene we wanted to express. We decided that we would have a BioBrick prefix and a 5mer followed by a sequence of nucleotides whose identity was up to us. Using PCR we would get the second double strand if we have the following:

BioBrick Prefix--ATG--5mer--gene until we get to restriction site

The PCR primer would attach to the ATG and the 5mer and would replicate the complementary base pairs to make a double stranded DNA sequence. As several of us were confused about PCR methods, we looked at the online link provided by Alyndria Thompson's link to the PCR interactive website which was very helpful. We also did some research to discover that the restriction enzyme BseRI (which is contained in the YFP that we wanted to alter) is not compatible with any other restriction enzyme. This is good because we did not want to plasmid containing this gene to close up with Xba1 once we digested it.

The team experienced some confusion with the hybrid idea and spoke to Clif Davis of Missouri Western University on the phone. He told us that there will be one part that is a fragment of the beginning of some nucleotides as a restriction cleaving site, ATG, the logical clause, and the start of the YFP gene that will be placed into a plasmid containing a space. To the left of the space will be the BioBrick Prefix and the right of the space will contain the rest of the genes in the YFP. Therefore, we decided not to alter any BioBrick ends and just look at restriction sites at the beginning of reporter proteins. The small fragment that will be placed into the plasmid will be made by PCR. The PCR will attach a primer onto the ATG and 5mer and replicate.

While we were discussing this, we expanded upon the idea that we can do whatever we want with the "suffix" portion between the 5mer and the beginning of YFP. We decided it would be best to remove ATG and add the first 5 amino acids and the restriction enzyme site. Along with this, if we have the 5mer coding for a certain amino acid (ie methionine) we could replace another codon that codes for methionine WITH that 5mer so long as this codon is contained within the first 15 nt. This way, we would not be altering the protein.

Olivia Ho-Shing came up with an idea of cascading PCR effect for longer logical clauses. Post translational modification of the protein was another one of her ideas. This applies because although we may code for the protein in a given way, the protein may modify or fix itself up to perform a slightly different function in its tertiary form.

Dr. Campbell suggested that we can look up restriction enzymes 400-500 base pairs into the reporter protein we would like to use; however, we have more flexibility in our procedure if we choose more "hardy" cutters. So instead, we could cut with EcoRI instead of with Xba1.

During the afternoon, we set about three tasks:
1) Which primers to use
2) Which promoters to use
3) Which restriction enzymes and how they will determine use of reporter proteins

Olivia Ho-Shing and I began looking up which restriction enzymes were strong so we could base our reporters on that. We began with Tet and RFP since those were the two reporters we had started out with choosing. I found a website that provided enzyme cleavage efficiency based upon the proximity of the site to the end of the PCR fragment that contains it. We discovered the following based upon the attached documents:

http://gcat.davidson.edu/GcatWiki/images/8/8f/Restriction_Enzyme_Cleavage_Efficiency.doc
http://gcat.davidson.edu/GcatWiki/images/9/9a/07Jun09_RE_Cutting_Efficiency_oh.doc

We decided that these two reporters contained efficient enzymes in a good quantity to choose from. On Monday, we will look through these enzymes once more and finalize which enzymes we would like to use with RFP and Tet.

==Sunday, June 7, 2009==

We did preparations for the mini prep this evening. The following notes are courtesy of Olivia Ho-Shing.

1. Sterically transfer ~2mL LB+AMP (AMP are ampicillin resistant plasmids) *Never put fluid back into bottle once it has entered pipette*
2. Use forceps to grab toothpick, scrap some frozen cells, rub in tube with top

'''Tubes'''

S03511 (2)

K091111

B0030

I715039

K091112

I715039

J31007 (2)

B0034

E1010

S03710

==Monday, June 8, 2009==

We reviewed the PCR process this morning as well as the restriction enzyme cleavage process.

Then, Leland Taylor, Olivia Ho-Shing and I all worked on converting the tRNA oligos we made to the template that Missouri Western used. The oligos we had previously made were different from the ones that Dr. Anderson had originally emailed us because further communication between Dr. Anderson and Missouri Western resulted in a better template.

We tried several times to minimize the number of oligos while keeping their individual lengths under 80bp. We managed to do this in the end by creating 7 oligos, 2 of which are variable between the suppressor tRNAs.
We reviewed the following document and finalized the oligos that we needed to order for the frameshift suppressor tRNAs. We decided to keep both the 10bp and 9bp anticodons of CCAUC.

I also looked up the amino acids that corresponded to the FS (frameshift suppressor) tRNAs for future use:

1. CUAGU = Leu
2. CCCUC = Pro
3. CGGUC = Arg
4. CCACU = Pro
5. CCAUC* = Pro
*9 and 10 bp anticodons

Also, we have decided to use NcoI as the restriction enzyme for RFP. This cleavage site occurs 419 bp into the gene. We have decided to use BamHI as the enzyme for Tetracycline Resistance and the cleavage site occurs 290bp into the gene. Both of these enzymes are very powerful and hardy.

We also looked over Missouri Western's tRNA oligos and primer oligos which we approved.
Our oligos were also approved by Missouri Western and Dr. Campbell ordered them while we supervised.

In the afternoon we did the miniprep. The mini prep directions were as appear in the link below.

http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_MiniPrep.html

We then worked with the Nano Prep to dilute our solution of DNA to an absorption of 1.5 and then we made labels for the tubes. The chart below shows relevant information we obtained from the NanoDrop for each of the reporter plasmids.

[[Image: Screen-capture-6.png]]

==Tuesday, June 9, 2009==

'''Preparations for restriction enzymes digestion of plasmids'''

Purpose: To determine the size of the plasmid inserts and ensure that plasmids are what we extracted from the cells during the mini-prep.

Things we need:
DNA ~3uL
Buffer 10x (Want to dilute to 1x) so add 2uL
Enzyme 1 ~1uL
Enzme 2 ~1uL
Water ~13uL
Final Volunter about 20 uL.

Need to choose the right buffer for the restriction enzymes. All buffers come in 10x (10 times more concentrated than what you want it to be when you use it). [http://gcat.davidson.edu/GcatWiki/index.php/Davidson_Missouri_W/Double_Digest_Guide Buffer Website]

Total volume of enzymes has to be 10% or less than final volume (most volume 2uL). Barely submerge the pipet tip into the solution when pipeting the enzymes.
Star (*) activity: Restriction enzyme stops cutting specifically and starts cutting everywhere.

DNA can't be seen by itself so we add EtBr (the bigger the insert, the more will bind) so bigger strands appear brighter. samples.

The order of entry doesn't matter, but make sure to enter the enzymes last.

Will have lots of labeled tubes and will put 3uL of appropriate plasmids into the tubes.
In the meantime, we'll make a "Master Mix" of all the other ingredients in a bigger tube. Always make enough volume for one extra tube.

Enzymes usually have an optimal temperature of 37C. We will let this reaction go for an hour and then we will run it on a gel.

'''Gel Electrophoresis Protocol'''

Website online for ideal gel concentration for resolving different sizes of molecules [http://gcat.davidson.edu/iGEM08/gelwebsite/gelwebsite.html Gel Concentration Website].
See protocol on the Davidson/Missouri Western Wookie for running molecules in the gel and how to digest with restriction enzymes.

'''What's Next?'''

1. 5' end additions
a. Using PCR
b. Cut at the restriction sites EcoR1 and either Bam H1 or Nco1
c. Cut plasmid and reporter with same restriction enzymes as above
d. Run a gel and purify "keeper" DNA (Ligate transform, screen, sequence)
e. Then, we take the "keeper" DNA and combine it with the insert from part

We can have steps d and e ready in advance.

2. tRNAs
a. Assemble the oligos into genes (Requires lots of calculations beforehand, but the actual steps )
b. Plasmids cut up with EcoR1 and Pst1 '''WE CAN USE THE "WASTE" PLASMID FROM THE RESTRICTION ENZYME DIGEST'''
c. Gel purify the plasmids (Ligate transform, screen, sequence)

The following tables include Master Mix measurements, agarose gel concentrations and buffers.
http://gcat.davidson.edu/GcatWiki/images/0/08/Shamitapic3.png
http://gcat.davidson.edu/GcatWiki/images/d/d1/Shamitapic4.png
http://gcat.davidson.edu/GcatWiki/images/6/6f/Shamitapic5.png

We made the Master Mix with our DNA and put it in the incubator at 10:40 AM.

'''Preparing the Gels'''

Now, we will be making 2 gels, 1 at 0.8% and 2.5% (weight to volume percent). The gel volume is 60mL.

'''Definition of 1% solution:
1 gram of agarose/ 100mL buffer'''

So for a 0.8% solution: 0.8 grams agarose/100mL buffer AND we need 60mL (fixed volume for casts run) so multiply that by 0.6.
Final concentration:

.48 grams agarose/60mL buffer

1 uL of EtBr stock for 60mL of gel.
EtBr is in the buffer so it is constantly replenishing itself even though it is moving toward one end in order to make bands on the other end visible.

After Dr. Campbell's demonstration, we each helped prepare a 0.8% buffer solution using the agarose powder, buffer and EtBr. We mixed together the first two substances in a 200mL Erlenmeyer flask and heated it for 120 seconds in the microwave. We then added EtBr and poured the gel solution into the gel cast.

'''Agar Plate'''

After lunch, we grew some more cells for each of the 11 parts in an agar plate labeled "LBAMP-AMC-9 June" so that so would have extra cells that could be stored for up to 2 weeks.
We made small circles on the agar plate and numbered them 1-11. The numbers corresponded to the following parts. We put the plate in the incubator overnight to let the cells grow.

1. B0030 AMC
2. K091111
3. S03710
4. I715039 OEH
5. S03511 ORH
6. I715039 AST
7. B0030 SDP
8. K091112
9. J31007 LJH
10. J31007 REC
11. S03511

'''Unloading the Gels'''

The following shows which parts we entered in which gels:

[[Image: screen-capture-2.png]]

We unloaded the 2.5% agarose first and after observing the bands under a UV light, put the gel into the BioRad machine to observe the bands. The image below is of a Molecular Weight Marker that shows the number of bp in control fragments of DNA according to the distance they travel down the gel.

[[Image: DNA_molecular_weight_marker_gel.png]]

We obtained the following printout for the first gel that was 2.5% Agarose.
[[Image: 2009-06-09_14hr_44min.jpg]]

The bands in the first well at the bottom are those of the molecular weight marker. We skipped the lane above (to the right) of the MWM and entered the rest of the inserts as described in the above table. A light band is visible for each of the 4 other wells. In Lanes 3 and 4 we concluded we are supposed to have approximately 45 bp because the insert is 15bp + 30 bp on either side of the insert (Xba1 and Spe1 restrictions sites + sticky ends + extra bases). We used the same logic for Lanes 5 and 6 which are supposed to contain approximately 86 base pairs.

One discrepancy in this gel, however, is that Lanes 5 and 6, which according to their labels respectively contain the LacIQ promoter and the Lac IQ1 promoter. According to the information online at the parts registry website, pLacIQ1 should be the same length as pLacIQ. The gels do not show this, but instead show that the sample in Lane 5 is smaller than that in Lane 6 because Lane 5 traveled further. Leland Taylor and Romina Clemente sent out an email to Pallavi Penumetcha to ask about this because she had written a paper about this.

We then unloaded the 0.8% agarose gel and then placed this in the BioRad machine under the same protocol as above. The following image is the 0.8% Agarose.

[[Image:2009-06-09_15hr_21min.jpg]]

We observed that the bands in Lane 1 (which is now at the top) are examples of high intensity bands. The plasmids contained in the original well are twice the intensity of the insert.

In Lane 5, however, we observed several different bands. This could be due to the extra DNA that was sitting on the top of the well. We decided to re-expose the gel to get better observations on Lanes 3 and 4 because the bands were not as intense. We anticipated seeing a band at about 250 bp because this was the size of the insert (plus extra bp). After re-exposing the gel, we did not find any difference and still did not see any bands in these lanes. We have decided to redo the gel on the part S03511 and so we prepared the master mix with that DNA and incubated it so that we could run the gel tomorrow.

We also cut out the "empty" plasmids from the gel so that we could use this as vectors for the coding sequence of the suppressor tRNAs.

Tomorrow we will be planning out specific tasks for people once the oligos arrive.

==Wednesday, June 10, 2009==

We began this morning by making the agarose gel with the same protocol as yesterday. This time, since we are just re-doing both S03511 inserts, we will have a 1.8% agarose gel.

'''OUTLINE: WHERE DO WE STAND?'''

Yesterday
a. Isolated empty plasmids
b. Verified most inserts
c. Waiting for:
>tRNA oligos
>Primers
a. Will amplify the reporter gene in the plasmid itself

Next?
a. When Primers Arrive
> Amplify the reporter genes with primers (PCR takes ~3 hours)
> Calculate how much reporter plasmid we will need (Start with 50x more than we actually will need)
> Cut amplified sequence (EcoR1 and BamH1/Nco1) then purify what we need
> Cut reporter plasmid (EcoR1 and BamH1/Nco1) then purify what we need
> Ligate amplified sequence and reporter plasmid
b. When tRNA oligos arrive
> Calculate how much of each oligo we will need (takes a while...)
> "Boil and cool" tRNA oligos together
> Ligate fragments (dsDNA) into plasmids we isolated yesterday (~5 minutes)
> Transform the plasmids (~20 minutes)

* LOOK THROUGH LIGATION PROTOCOL (How to assemble genes)
* LOOK THROUGH TRANSFORMATION PROTOCOL

''What we need to get done today'':
1. Prepare the control for the Tet Resistance by BioBricking the part pLac+RBS (S03511) with the TetA (J31007).
2. Check the part number for RFP and also for pLac+RBS+RFP
3. Read through ligation protocol
4. See how many nanograms of plasmid we will need for the tRNAs and for the inserts and multiply this number by 50.
> Check if we have enough plasmids from our mini-prep
5. Purify the plasmids from the gel

After we made this outline, Leland, Olivia and I analyzed the gel we ran this morning and did not see a more than one band in the 2 lanes that contained DNA from S03511 (image below). Since we did not see multiple bands in yesterday's gel either, we conclude that there is something wrong with the DNA rather than with the restriction enzymes that we used to cut it. We concluded that there is probably not an insert in this plasmid both the part and the plasmid are useless.

[[Image: 2009-06-10 10hr 30min.jpg]]

Now, we do not have an immediately accessible promoter. We have decided to order the pBAD promoter from Missouri Western and we will attach the RBS+Tet to this plasmid. Our insert for this will be the larger gene because it is easier to manipulate in the gel than the small gene.

Alyndria Thompson and Romina Clemente are figuring out the size of the plasmids including the reporter genes.

After lunch we performed several steps to isolate and purify the gel to get the empty plasmids will be the vectors for the tRNA DNA. Then, we used the NanoDrop to assess the purity of our DNA. We found that the plasmids were more pure than we had expected. The following values were obtained:

Abs 1.850
A-260 10 mm path 0.172
A-280 10 mm path 0.075
260/280 2.28
260/230 0.09

We then followed the protocol for preparing the agar plates. We put the plate with the agar solution in the Autoclave.

While the solution was in the autoclave, we discussed that we don't really need a promoter in front of RBS+Tet for the gene to be expressed because Tet expression is so strong that the gene would translate without a promoter. However, it would be a good idea to have the promoter because if it was possible to reinforce the translation of the reporter protein, we wanted to do that.

We found that there were some tubes in the lab fridge that contained the pBAD and pLac promoters with RBS. We decided to use these as the promoter and RBS for our experiments. The pLac promoter will be used for RFP and the pBad promoter used with Tet Resistance. We decided to use the pBad with Tet because it is weaker in comparison to pLac. As expression of the Tet gene can be toxic to a cell, we do not want to overexpress the gene with a stronger promoter.

Once the agar solution was sterilized in the Autoclave, we removed it, cooled it, and pipetted the antibiotic resistance into it. We then poured the solution into the plates, removed any bubbles, and let the gel form.

Alyndria Thompson and Romina Clemente concluded that we had more than enough recieving vectors for both the RFP and Tetracycline genes. Since we need 50 nanograms, it is wise to have at least 50 times that much that we can work with, so we need at least 2,500 ng.

RFP 1: 77.6 x 4 = 310.4 ng/uL
x 20 uL = 3,272 ng
RFP 2: 59.5 x 4 = 238 ng/uL
x 20 uL = 4,760 ng
Tet: 40.9 x 4 = 163.6 ng/uL
x 20 uL = 3,272 ng

According to these calculations we have more than 50 times what we need for both instances of RFP and Tet.

Tomorrow, we can prepare the Master Mix for the digestions of both reporter plamids and then add the restriction enzymes once they arrive. Then, we can follow through with the rest of the plans we had made above in the section titles "Next?".

==Thursday, June 11, 2009==

We began this morning by calculating how many uL we would need for each of the mixes for digesting our reporter plasmids. We decided to double the final volume of each mix to 40uL instead of 20uL. Since we knew the concentration of each plasmid and also knew we needed 2500 ng, we cross multiplied to find the uL of DNA we would need for each.

Concentration Calculations:
310.4 ng/1 uL = 2500ng/x uL
x = 8.05 uL

Here is a chart indicating how much of each we used for the 2 mixes.

[[Image: DoubledigestRFPTetR.png]]

On the Promega website, we found a list of compatible buffers for double digestions. Buffers E and H are compatible for the Tet plasmid and RFP plasmid digestions respectively. We fortunately have both buffers in stock.

[[Image:BamEcoR1.png]] [[Image:NcoEcoR1.png]]

We waited for our waited for the restriction enzymes to arrive and in the meantime, we transformed the plasmids with pLac+RBS, pBad, and pBad+RBS into ''E.coli'' cells. We want the ''E.coli'' cells to replicate the plasmids with these genes because we have a limited amount of genes. [http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_Transformation.html Zippy Plasmid Transformation]

Once our oligos and restriction enzymes arrived, we got to work right away!

'''PCR (Polymerase Chain Reaction)'''

We calculated how much plasmid we will need for the PCR. We decided to dilute the concentration of our plasmids a hundred fold so that we could get pipetable amounts.

''Change in Concentrations''
310.4 ng/uL x 1/100 (dilution factor) = 3.104 ng/uL (RFP)
163.6 ng/uL x 1/100 (dilution factor) = 0.164 ng/uL (Tet)

''Example shown with RFP''
3.104 ng/1 uL = 1ng/ x uL, x = .32
For Tet, x = .61

We also calculated how much of each primer we would need with the following equation:

C1V1 = C2V2
(100 uM) (x) = (1 uM)( 100 uL)
x = 1 uL

[[Image: screen-capture-8.png]]

Each of the 5 of us made had 2 tubes (one for each reporter protein) because there were 5 different forward primers.To each tube, we added the template DNA (either RFP or Tet), water, the respective reverse primer (either RFP or Tet), and the forward primer depending on which 5mer we had chosen. My 5mer was CCAUC. Finally, we added the Master Mix last because this contained the enzyme (DNA polymerase) that would amplify our desired sequence.

[[Image: Pcrtubes.png]]

We followed the temperature cycles so that PCR could occur. The annealing temperature needed to be 5C less than the smallest melting temperature of the primers. Since the reverse primer of RFP had a melting temp of 66C, we set the temperature of the 3rd cycle on 61C.

[[Image: Temperaturecycle.png]]

'''tRNA Oligo Assembly'''

We needed to calculate the concentration of each of the oligo before carrying out the assembly. The stock concentration of each of the oligo was 100 uM. Since we wanted to have 5uM of the oligo in the test tube, and we know that final volume of the test tube is 20 uL, we just cross multiplied to find the amount we needed to add to the tubes to get this concentration.

(100uM)x = (5uM)(20uL)
x = 1 uL

We added 1 uL of each oligo to the tubes.

We had six total tRNAs we needed to assemble so each of us took one FML (one person took two) and to each tube we added 1uL of each oligo for a total of 7uL, 2uL of buffer, and 11uL of water for a total volume of 20 uL. Five of the oligos were constant and 2 varied for each of the different tRNAs. So everyone pipetted the same 5 oligos and everyone also had 2 unique oligos to add. When all of the oligos were added and the tubes ready, we boiled the tubes at 50C for 5 minutes. We then allowed the tubes to remain in the water to cool very slowly overnight so that the annealing process could take place.

The last thing we did at the end of the day was finish off our plasmid digestion that we had begun in the morning. We added the buffer, DNA, water, and restriction enzymes and placed this in the incubator for overnight digestion.

'''To Do''':

1. Isolate our desired dsDNA from the PCR.
2. Verify our tRNA inserts and eventually transform these into plasmids.
3. Check our agar plates to see colonies that have replicated our pLac+RBS, pBAD+RBS, and pBAD genes.

==Friday, June 12, 2009==

This morning, we began by outlining the main items on today's agenda.

1. tRNAs: Ligation, Transformation
2. Digestion reporters (run all of it and gel purify)
3. Transformed cells
4. PCR: Gel run an aliquot to verify (5uL)

We used the equation given by the Ligation protocol to calculate how many nanograms of insert we would need. Initially, we started out calculated the MW average of the 9 and 10 anticodon loops of tRNAs which were 15828.23 ug/umols and 159016.6 ug/umols respectively. We then saw, however, that these would not be helpful values in the process. Instead we started out with the equation below to find the final ng of insert we would need for the ligation.

[[Image: ligationcalculations.png]]

We calculated our initial concentration in ng/uL of insert as shown below.

[[Image: ligationequation.png]]
X nanograms of insert = [(2)(144bp)(50 ng linearalized plasmid)]/2038bp
X = 7.0657 ng of insert

We wanted to find the amount of uL of insert we would need to use. We diluted the initial concentration 50x because we wanted a pipettable amount of insert. Since we knew we wanted the final amount of nanograms to be 7.0657, we calculated as shown below:

''DILUTION'': 555.48/50 = 11.012
''CALCULATION'': 11.012 ng/ 1uL = 7.065 ng/ x uL
x = .64 uL of insert

After we had done these calculations, half of the group started to carryout the ligation of the tRNA inserts into empty plasmids. We needed 7 ligations total: 6 were the tRNAs we needed, while the last one is a negative control. These are the final amounts of what we used:

[[Image: Trnaligation.png]]

The other half of the group (which included me) prepared the agarose gels so that we could run gels for the digested reporters and the PCR inserts. We calculated that the digested reporters needed an agarose of 4% and the PCR reporters needed an agarose of 1.45%. We allowed this gel to form for about 45 minutes to an hour. We prepared both the gels and allowed them to run through lunch.
After lunch, we took pictures of our gels and noticed that the third lane in the PCR gel (third lane from the top in the bottom photo) did not develop a band. The lane was supposed to contain the CUAGU insert for Tetracycline Resistance.

[[Image: 2009-06-12 15hr 54min.jpg]] [[Image:2009-06-12_16hr_07min.jpg]]

Romina Clemente and I decided to repeat this PCR using the TetResistance and RBS+TetResistance plasmids as templates. We had to recalculate how much template DNA to use. We let the PCR run overnight. We also verified that the digestion fragments (both the plasmid and the insert) on the other gel were the correct size. The plasmids, which are the brighter fragment in the gel with only 2 lanes, contain the plasmid base pairs along with the rest of our reporter genes. We isolated these plasmids from the gel today and stored them for purification on Monday.

In the meantime, other members of our lab had finished the ligation of the tRNA genes to the empty plasmids and were now transforming these into competent cells. They placed the agar plate with these cells in the fridge overnight.

We also observed the transformations that we had performed yesterday and noticed that I13453 did not form any colonies. J04500 and J3102 did form colonies.

To Do:
1. PCR: Dig, Ligate, Transform
Clean and concentration column, digest, clean and concentration column, ligate.
2. Gel purification of our digestion plasmids
3. Check if colonies have grown for our tRNAs

File:DNA molecular weight marker gel.png

2009-06-14T22:03:04Z

ShPunjabi:

IGEM Notebook

2009-06-12T23:03:08Z

ShPunjabi: /* Friday, June 12, 2009 */

==Wednesday, June 3, 2009==

Romina Clemente and I are trying to find suitable reporter proteins to use. Yesterday, Leland Taylor and Alyndria Thompson were working on ways to insert the gene sequences into the plasmid. Upon seeing how they wanted to manipulated the reporter gene to include the logical clauses, we came up with a few criteria for the reporter genes we would use. The following criteria for genes are listed in order of the broadest aspect to look at to the narrowest aspect:
a) Doesn't contain restriction sites for the 4 restriction enzymes (EcoR1, Xbal, Spel, Pst1) used to cleave the Biobrick part out of the plasmid.
b) Contains 6 cutter restriction sites.
c) These restriction enzymes aren't blunt (cleave straight down at one spot).
d) These restriction sites are close to thge 5' (beginning) end of the sequence.
e) These enzymes are easiest to work with and cheapest.
We are finding the part numbers of the reporter genes we want to use (antibiotic resistance, fluorescence, LacZ) through our own GCAT because we know these ones work. We are then locating these parts on the parts registry [1] website. We copied and pasted the gene sequences we obtained from the registry onto the ApE software [2]. From here, we were able to generate a genetic map of each gene that outlined each restriction site that fit our criteria. We put each genetic map, alongside the part number used, into a Word document.

http://gcat.davidson.edu/GcatWiki/images/0/0e/Restriction_Site_Mapping_on_Reporter_Genes.doc

Later on in the day, we decided that the best way to test for suppression would involve placing the 5mers at the beginning of the reporter gene instead of inserting them into the reporter gene. To do this, we decided that the gene along with its start codon need to be expressed after the 5mer. We will insert a BioBrick plasmid with a 5mer and start codon that will be incorporated before the gene:
RBS-6-8nt-ATG-5mer--ATG-gene

Leland Taylor assembled the oligos that we will need for the BioBrick pieces.

In order to test whether the reporter protein would be expressed regardless of the 5mer being suppressed or not, we would need to remove the suppressor tRNAs from the cell.

Meanwhile, I helped figure out the coding sequence that we will need for the suppressor tRNAs that will bind to the 5mers. We decided that we would be using the following suppressor tRNAs on the Davidson side: CUAGU, CCCUC, CGGUC, CCAUC, and CCACU. We searched through several papers, with little result and finally emailed Dr. Christopher Anderson of UC Berkeley to request the DNA sequence. He emailed us back with a generic DNA code that we could use to create the tRNA; however, for each suppressor tRNA we would use, we would need to change base pairs in the anticodon loop.

The length of the tRNA is 92 nt. The anticodon loop is comprised of 9 nt. Once we have BioBricked the DNA sequence with 4 restriction sites and supplemental nucleotides, the length of the entire gene is 144 nucleotides. This is made up of the 92 nt +22 nt on each side of the sequence (44) + 4 nt on each side of the restriction ends (8).

At the end of the day, we decided to use tetracycline resistance and RFP as our reporter proteins for this tester experiment.

==Thursday, June 4, 2009==

This morning, I began the day by learning how to use the PEARL prgram. Olivia Ho-Shing sat with me and walked me through the program script. And then, Leland Taylor and Shashank Suresh had a problem they wanted us to solve. Leland explained that we need to make sure there is a stop codon in the gene if we fail to suppress the 5mer. The stop codon needs to be before we find another 5mer. They also wanted us to run through both the RFP and Tet Resistance genes to make sure that they did not contain any of the anticodon sequences from our 5 frameshift suppressor tRNAs so as to throw the translation out of frame. They further wanted us to confirm that if we do have successful suppression, there are no stop codons in the middle of the RFP and Tet Resistance genes. PEARL did not find any matches for the suppressor tRNAs within genes. We found 3 matches for stop codons UAA. Two of these occured at the end of the Tet and one at the end of RFP, where they should normally be to end translation into a protein.

Following this, Olivia Ho-Shing and I tried to determine how we can add BioBrick ends to our single stranded DNA sequences that code for the suppressor tRNAs. Our ultimate goal is to find complements for these single strands so they can be put into plasmids. We found the standard prefixes and suffixes for sequences that do not contain ATG (becuase this sequence is a functional RNA and will not be translated) and we placed these before and after the altered 92bp sequence that Dr. Anderson provided us with. The prefixes and suffixes are necessary because they are the extra nucleotides that will be mimicking a "restriction enzyme cutting site". The single strand appeared as below after the Bio Brick prefixes and suffixes were added:

22ntPREFIX--32nt--VARIABLE ANTICODON LOOP--51nt--21ntSUFFIX

We used the suppressor codon CUAGU as our example and placed its appropriate anticodon loop in the sequence. We put this into the lancelator that would give us the other strand as well as the oligos we would need for ideal construction of this double stranded DNA fragment to put into the plasmid. We recieved a total of 4 oligos, 2 of which were variable and 2 of which were constant for all 6 different suppressor tRNAs. Using this double strand, we simulated a restriction enzyme digestion by EcoRI and PstI. We took off the G at both ends of the top strand and then removed the first five nucleotides on each end of the bottom strand. This made the top strand longer than the bottom one. We then proceeded to take the two variable strands and changed the anticodon loop nt for each suppressor tRNA we used.

Around lunchtime, we encountered a problem with the BioBrick scar in the "ATG-5mer subpiece" of the test project. It turns out that after annealing the sticky ends of the Xba1 and Spe1 sites, the scar created had a stop codon TAG (UAG) that was in frame with the suppressed codon. When the Xba1 site on insert anneals to the Spe1 site on the plasmid (the green portions), we get a scar that reads TACTAG.
[[Image:screen-capture.png]]

For the afternoon, Olivia Ho-Shing and I finished assembly of the oligos of the suppressor tRNA codes. We assembled the document below:

http://gcat.davidson.edu/GcatWiki/images/5/5e/TRNAoligostoOrder.doc

We were assigned tasks of creating "cartoons" to illustrate the phenomenon that had occurred. No one could successfully do this however, because we could not understand how the piece had formed. The parts registry website provided wrong information on the restriction site of Spe1 which slowed our efficiency. This project turned into an effort to try to find two other different restriction enzymes that have complementary sticky ends other than Xba1 and Spe1. We want to get rid oc the scar that contains TACTAG.

We ended up deciding that a hybrid of two ideas was needed: restriction enzymes would need to be used to make several reporter gene sequences with different beginnings dependending on the suppressor tRNA sequence needed.

==Friday, June 5, 2009==

Our goal for this morning was to discuss the hybrid PCR approach to assemble the part that comes before the gene we wanted to express. We decided that we would have a BioBrick prefix and a 5mer followed by a sequence of nucleotides whose identity was up to us. Using PCR we would get the second double strand if we have the following:

BioBrick Prefix--ATG--5mer--gene until we get to restriction site

The PCR primer would attach to the ATG and the 5mer and would replicate the complementary base pairs to make a double stranded DNA sequence. As several of us were confused about PCR methods, we looked at the online link provided by Alyndria Thompson's link to the PCR interactive website which was very helpful. We also did some research to discover that the restriction enzyme BseRI (which is contained in the YFP that we wanted to alter) is not compatible with any other restriction enzyme. This is good because we did not want to plasmid containing this gene to close up with Xba1 once we digested it.

The team experienced some confusion with the hybrid idea and spoke to Clif Davis of Missouri Western University on the phone. He told us that there will be one part that is a fragment of the beginning of some nucleotides as a restriction cleaving site, ATG, the logical clause, and the start of the YFP gene that will be placed into a plasmid containing a space. To the left of the space will be the BioBrick Prefix and the right of the space will contain the rest of the genes in the YFP. Therefore, we decided not to alter any BioBrick ends and just look at restriction sites at the beginning of reporter proteins. The small fragment that will be placed into the plasmid will be made by PCR. The PCR will attach a primer onto the ATG and 5mer and replicate.

While we were discussing this, we expanded upon the idea that we can do whatever we want with the "suffix" portion between the 5mer and the beginning of YFP. We decided it would be best to remove ATG and add the first 5 amino acids and the restriction enzyme site. Along with this, if we have the 5mer coding for a certain amino acid (ie methionine) we could replace another codon that codes for methionine WITH that 5mer so long as this codon is contained within the first 15 nt. This way, we would not be altering the protein.

Olivia Ho-Shing came up with an idea of cascading PCR effect for longer logical clauses. Post translational modification of the protein was another one of her ideas. This applies because although we may code for the protein in a given way, the protein may modify or fix itself up to perform a slightly different function in its tertiary form.

Dr. Campbell suggested that we can look up restriction enzymes 400-500 base pairs into the reporter protein we would like to use; however, we have more flexibility in our procedure if we choose more "hardy" cutters. So instead, we could cut with EcoRI instead of with Xba1.

During the afternoon, we set about three tasks:
1) Which primers to use
2) Which promoters to use
3) Which restriction enzymes and how they will determine use of reporter proteins

Olivia Ho-Shing and I began looking up which restriction enzymes were strong so we could base our reporters on that. We began with Tet and RFP since those were the two reporters we had started out with choosing. I found a website that provided enzyme cleavage efficiency based upon the proximity of the site to the end of the PCR fragment that contains it. We discovered the following based upon the attached documents:

http://gcat.davidson.edu/GcatWiki/images/8/8f/Restriction_Enzyme_Cleavage_Efficiency.doc
http://gcat.davidson.edu/GcatWiki/images/9/9a/07Jun09_RE_Cutting_Efficiency_oh.doc

We decided that these two reporters contained efficient enzymes in a good quantity to choose from. On Monday, we will look through these enzymes once more and finalize which enzymes we would like to use with RFP and Tet.

==Sunday, June 7, 2009==

We did preparations for the mini prep this evening. The following notes are courtesy of Olivia Ho-Shing.

1. Sterically transfer ~2mL LB+AMP (AMP are ampicillin resistant plasmids) *Never put fluid back into bottle once it has entered pipette*
2. Use forceps to grab toothpick, scrap some frozen cells, rub in tube with top

'''Tubes'''

S03511 (2)

K091111

B0030

I715039

K091112

I715039

J31007 (2)

B0034

E1010

S03710

==Monday, June 8, 2009==

We reviewed the PCR process this morning as well as the restriction enzyme cleavage process.

Then, Leland Taylor, Olivia Ho-Shing and I all worked on converting the tRNA oligos we made to the template that Missouri Western used. The oligos we had previously made were different from the ones that Dr. Anderson had originally emailed us because further communication between Dr. Anderson and Missouri Western resulted in a better template.

We tried several times to minimize the number of oligos while keeping their individual lengths under 80bp. We managed to do this in the end by creating 7 oligos, 2 of which are variable between the suppressor tRNAs.
We reviewed the following document and finalized the oligos that we needed to order for the frameshift suppressor tRNAs. We decided to keep both the 10bp and 9bp anticodons of CCAUC.

I also looked up the amino acids that corresponded to the FS (frameshift suppressor) tRNAs for future use:

1. CUAGU = Leu
2. CCCUC = Pro
3. CGGUC = Arg
4. CCACU = Pro
5. CCAUC* = Pro
*9 and 10 bp anticodons

Also, we have decided to use NcoI as the restriction enzyme for RFP. This cleavage site occurs 419 bp into the gene. We have decided to use BamHI as the enzyme for Tetracycline Resistance and the cleavage site occurs 290bp into the gene. Both of these enzymes are very powerful and hardy.

We also looked over Missouri Western's tRNA oligos and primer oligos which we approved.
Our oligos were also approved by Missouri Western and Dr. Campbell ordered them while we supervised.

In the afternoon we did the miniprep. The mini prep directions were as appear in the link below.

http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_MiniPrep.html

We then worked with the Nano Prep to dilute our solution of DNA to an absorption of 1.5 and then we made labels for the tubes. The chart below shows relevant information we obtained from the NanoDrop for each of the reporter plasmids.

[[Image: Screen-capture-6.png]]

==Tuesday, June 9, 2009==

'''Preparations for restriction enzymes digestion of plasmids'''

Purpose: To determine the size of the plasmid inserts and ensure that plasmids are what we extracted from the cells during the mini-prep.

Things we need:
DNA ~3uL
Buffer 10x (Want to dilute to 1x) so add 2uL
Enzyme 1 ~1uL
Enzme 2 ~1uL
Water ~13uL
Final Volunter about 20 uL.

Need to choose the right buffer for the restriction enzymes. All buffers come in 10x (10 times more concentrated than what you want it to be when you use it). [http://gcat.davidson.edu/GcatWiki/index.php/Davidson_Missouri_W/Double_Digest_Guide Buffer Website]

Total volume of enzymes has to be 10% or less than final volume (most volume 2uL). Barely submerge the pipet tip into the solution when pipeting the enzymes.
Star (*) activity: Restriction enzyme stops cutting specifically and starts cutting everywhere.

DNA can't be seen by itself so we add EtBr (the bigger the insert, the more will bind) so bigger strands appear brighter. samples.

The order of entry doesn't matter, but make sure to enter the enzymes last.

Will have lots of labeled tubes and will put 3uL of appropriate plasmids into the tubes.
In the meantime, we'll make a "Master Mix" of all the other ingredients in a bigger tube. Always make enough volume for one extra tube.

Enzymes usually have an optimal temperature of 37C. We will let this reaction go for an hour and then we will run it on a gel.

'''Gel Electrophoresis Protocol'''

Website online for ideal gel concentration for resolving different sizes of molecules [http://gcat.davidson.edu/iGEM08/gelwebsite/gelwebsite.html Gel Concentration Website].
See protocol on the Davidson/Missouri Western Wookie for running molecules in the gel and how to digest with restriction enzymes.

'''What's Next?'''

1. 5' end additions
a. Using PCR
b. Cut at the restriction sites EcoR1 and either Bam H1 or Nco1
c. Cut plasmid and reporter with same restriction enzymes as above
d. Run a gel and purify "keeper" DNA (Ligate transform, screen, sequence)
e. Then, we take the "keeper" DNA and combine it with the insert from part

We can have steps d and e ready in advance.

2. tRNAs
a. Assemble the oligos into genes (Requires lots of calculations beforehand, but the actual steps )
b. Plasmids cut up with EcoR1 and Pst1 '''WE CAN USE THE "WASTE" PLASMID FROM THE RESTRICTION ENZYME DIGEST'''
c. Gel purify the plasmids (Ligate transform, screen, sequence)

The following tables include Master Mix measurements, agarose gel concentrations and buffers.
http://gcat.davidson.edu/GcatWiki/images/0/08/Shamitapic3.png
http://gcat.davidson.edu/GcatWiki/images/d/d1/Shamitapic4.png
http://gcat.davidson.edu/GcatWiki/images/6/6f/Shamitapic5.png

We made the Master Mix with our DNA and put it in the incubator at 10:40 AM.

'''Preparing the Gels'''

Now, we will be making 2 gels, 1 at 0.8% and 2.5% (weight to volume percent). The gel volume is 60mL.

'''Definition of 1% solution:
1 gram of agarose/ 100mL buffer'''

So for a 0.8% solution: 0.8 grams agarose/100mL buffer AND we need 60mL (fixed volume for casts run) so multiply that by 0.6.
Final concentration:

.48 grams agarose/60mL buffer

1 uL of EtBr stock for 60mL of gel.
EtBr is in the buffer so it is constantly replenishing itself even though it is moving toward one end in order to make bands on the other end visible.

After Dr. Campbell's demonstration, we each helped prepare a 0.8% buffer solution using the agarose powder, buffer and EtBr. We mixed together the first two substances in a 200mL Erlenmeyer flask and heated it for 120 seconds in the microwave. We then added EtBr and poured the gel solution into the gel cast.

'''Agar Plate'''

After lunch, we grew some more cells for each of the 11 parts in an agar plate labeled "LBAMP-AMC-9 June" so that so would have extra cells that could be stored for up to 2 weeks.
We made small circles on the agar plate and numbered them 1-11. The numbers corresponded to the following parts. We put the plate in the incubator overnight to let the cells grow.

1. B0030 AMC
2. K091111
3. S03710
4. I715039 OEH
5. S03511 ORH
6. I715039 AST
7. B0030 SDP
8. K091112
9. J31007 LJH
10. J31007 REC
11. S03511

'''Unloading the Gels'''

The following shows which parts we entered in which gels:

[[Image: screen-capture-2.png]]

We unloaded the 2.5% agarose first and after observing the bands under a UV light, put the gel into the BioRad machine to observe the bands. The image below is of a Molecular Weight Marker that shows the number of bp in control fragments of DNA according to the distance they travel down the gel.

[[Image: screen-capture-1.png]]

We obtained the following printout for the first gel that was 2.5% Agarose.
[[Image: 2009-06-09_14hr_44min.jpg]]

The bands in the first well at the bottom are those of the molecular weight marker. We skipped the lane above (to the right) of the MWM and entered the rest of the inserts as described in the above table. A light band is visible for each of the 4 other wells. In Lanes 3 and 4 we concluded we are supposed to have approximately 45 bp because the insert is 15bp + 30 bp on either side of the insert (Xba1 and Spe1 restrictions sites + sticky ends + extra bases). We used the same logic for Lanes 5 and 6 which are supposed to contain approximately 86 base pairs.

One discrepancy in this gel, however, is that Lanes 5 and 6, which according to their labels respectively contain the LacIQ promoter and the Lac IQ1 promoter. According to the information online at the parts registry website, pLacIQ1 should be the same length as pLacIQ. The gels do not show this, but instead show that the sample in Lane 5 is smaller than that in Lane 6 because Lane 5 traveled further. Leland Taylor and Romina Clemente sent out an email to Pallavi Penumetcha to ask about this because she had written a paper about this.

We then unloaded the 0.8% agarose gel and then placed this in the BioRad machine under the same protocol as above. The following image is the 0.8% Agarose.

[[Image:2009-06-09_15hr_21min.jpg]]

We observed that the bands in Lane 1 (which is now at the top) are examples of high intensity bands. The plasmids contained in the original well are twice the intensity of the insert.

In Lane 5, however, we observed several different bands. This could be due to the extra DNA that was sitting on the top of the well. We decided to re-expose the gel to get better observations on Lanes 3 and 4 because the bands were not as intense. We anticipated seeing a band at about 250 bp because this was the size of the insert (plus extra bp). After re-exposing the gel, we did not find any difference and still did not see any bands in these lanes. We have decided to redo the gel on the part S03511 and so we prepared the master mix with that DNA and incubated it so that we could run the gel tomorrow.

We also cut out the "empty" plasmids from the gel so that we could use this as vectors for the coding sequence of the suppressor tRNAs.

Tomorrow we will be planning out specific tasks for people once the oligos arrive.

==Wednesday, June 10, 2009==

We began this morning by making the agarose gel with the same protocol as yesterday. This time, since we are just re-doing both S03511 inserts, we will have a 1.8% agarose gel.

'''OUTLINE: WHERE DO WE STAND?'''

Yesterday
a. Isolated empty plasmids
b. Verified most inserts
c. Waiting for:
>tRNA oligos
>Primers
a. Will amplify the reporter gene in the plasmid itself

Next?
a. When Primers Arrive
> Amplify the reporter genes with primers (PCR takes ~3 hours)
> Calculate how much reporter plasmid we will need (Start with 50x more than we actually will need)
> Cut amplified sequence (EcoR1 and BamH1/Nco1) then purify what we need
> Cut reporter plasmid (EcoR1 and BamH1/Nco1) then purify what we need
> Ligate amplified sequence and reporter plasmid
b. When tRNA oligos arrive
> Calculate how much of each oligo we will need (takes a while...)
> "Boil and cool" tRNA oligos together
> Ligate fragments (dsDNA) into plasmids we isolated yesterday (~5 minutes)
> Transform the plasmids (~20 minutes)

* LOOK THROUGH LIGATION PROTOCOL (How to assemble genes)
* LOOK THROUGH TRANSFORMATION PROTOCOL

''What we need to get done today'':
1. Prepare the control for the Tet Resistance by BioBricking the part pLac+RBS (S03511) with the TetA (J31007).
2. Check the part number for RFP and also for pLac+RBS+RFP
3. Read through ligation protocol
4. See how many nanograms of plasmid we will need for the tRNAs and for the inserts and multiply this number by 50.
> Check if we have enough plasmids from our mini-prep
5. Purify the plasmids from the gel

After we made this outline, Leland, Olivia and I analyzed the gel we ran this morning and did not see a more than one band in the 2 lanes that contained DNA from S03511 (image below). Since we did not see multiple bands in yesterday's gel either, we conclude that there is something wrong with the DNA rather than with the restriction enzymes that we used to cut it. We concluded that there is probably not an insert in this plasmid both the part and the plasmid are useless.

[[Image: 2009-06-10 10hr 30min.jpg]]

Now, we do not have an immediately accessible promoter. We have decided to order the pBAD promoter from Missouri Western and we will attach the RBS+Tet to this plasmid. Our insert for this will be the larger gene because it is easier to manipulate in the gel than the small gene.

Alyndria Thompson and Romina Clemente are figuring out the size of the plasmids including the reporter genes.

After lunch we performed several steps to isolate and purify the gel to get the empty plasmids will be the vectors for the tRNA DNA. Then, we used the NanoDrop to assess the purity of our DNA. We found that the plasmids were more pure than we had expected. The following values were obtained:

Abs 1.850
A-260 10 mm path 0.172
A-280 10 mm path 0.075
260/280 2.28
260/230 0.09

We then followed the protocol for preparing the agar plates. We put the plate with the agar solution in the Autoclave.

While the solution was in the autoclave, we discussed that we don't really need a promoter in front of RBS+Tet for the gene to be expressed because Tet expression is so strong that the gene would translate without a promoter. However, it would be a good idea to have the promoter because if it was possible to reinforce the translation of the reporter protein, we wanted to do that.

We found that there were some tubes in the lab fridge that contained the pBAD and pLac promoters with RBS. We decided to use these as the promoter and RBS for our experiments. The pLac promoter will be used for RFP and the pBad promoter used with Tet Resistance. We decided to use the pBad with Tet because it is weaker in comparison to pLac. As expression of the Tet gene can be toxic to a cell, we do not want to overexpress the gene with a stronger promoter.

Once the agar solution was sterilized in the Autoclave, we removed it, cooled it, and pipetted the antibiotic resistance into it. We then poured the solution into the plates, removed any bubbles, and let the gel form.

Alyndria Thompson and Romina Clemente concluded that we had more than enough recieving vectors for both the RFP and Tetracycline genes. Since we need 50 nanograms, it is wise to have at least 50 times that much that we can work with, so we need at least 2,500 ng.

RFP 1: 77.6 x 4 = 310.4 ng/uL
x 20 uL = 3,272 ng
RFP 2: 59.5 x 4 = 238 ng/uL
x 20 uL = 4,760 ng
Tet: 40.9 x 4 = 163.6 ng/uL
x 20 uL = 3,272 ng

According to these calculations we have more than 50 times what we need for both instances of RFP and Tet.

Tomorrow, we can prepare the Master Mix for the digestions of both reporter plamids and then add the restriction enzymes once they arrive. Then, we can follow through with the rest of the plans we had made above in the section titles "Next?".

==Thursday, June 11, 2009==

We began this morning by calculating how many uL we would need for each of the mixes for digesting our reporter plasmids. We decided to double the final volume of each mix to 40uL instead of 20uL. Since we knew the concentration of each plasmid and also knew we needed 2500 ng, we cross multiplied to find the uL of DNA we would need for each.

Concentration Calculations:
310.4 ng/1 uL = 2500ng/x uL
x = 8.05 uL

Here is a chart indicating how much of each we used for the 2 mixes.

[[Image: DoubledigestRFPTetR.png]]

On the Promega website, we found a list of compatible buffers for double digestions. Buffers E and H are compatible for the Tet plasmid and RFP plasmid digestions respectively. We fortunately have both buffers in stock.

[[Image:BamEcoR1.png]] [[Image:NcoEcoR1.png]]

We waited for our waited for the restriction enzymes to arrive and in the meantime, we transformed the plasmids with pLac+RBS, pBad, and pBad+RBS into ''E.coli'' cells. We want the ''E.coli'' cells to replicate the plasmids with these genes because we have a limited amount of genes. [http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_Transformation.html Zippy Plasmid Transformation]

Once our oligos and restriction enzymes arrived, we got to work right away!

'''PCR (Polymerase Chain Reaction)'''

We calculated how much plasmid we will need for the PCR. We decided to dilute the concentration of our plasmids a hundred fold so that we could get pipetable amounts.

''Change in Concentrations''
310.4 ng/uL x 1/100 (dilution factor) = 3.104 ng/uL (RFP)
163.6 ng/uL x 1/100 (dilution factor) = 0.164 ng/uL (Tet)

''Example shown with RFP''
3.104 ng/1 uL = 1ng/ x uL, x = .32
For Tet, x = .61

We also calculated how much of each primer we would need with the following equation:

C1V1 = C2V2
(100 uM) (x) = (1 uM)( 100 uL)
x = 1 uL

[[Image: screen-capture-8.png]]

Each of the 5 of us made had 2 tubes (one for each reporter protein) because there were 5 different forward primers.To each tube, we added the template DNA (either RFP or Tet), water, the respective reverse primer (either RFP or Tet), and the forward primer depending on which 5mer we had chosen. My 5mer was CCAUC. Finally, we added the Master Mix last because this contained the enzyme (DNA polymerase) that would amplify our desired sequence.

[[Image: Pcrtubes.png]]

We followed the temperature cycles so that PCR could occur. The annealing temperature needed to be 5C less than the smallest melting temperature of the primers. Since the reverse primer of RFP had a melting temp of 66C, we set the temperature of the 3rd cycle on 61C.

[[Image: Temperaturecycle.png]]

'''tRNA Oligo Assembly'''

We needed to calculate the concentration of each of the oligo before carrying out the assembly. The stock concentration of each of the oligo was 100 uM. Since we wanted to have 5uM of the oligo in the test tube, and we know that final volume of the test tube is 20 uL, we just cross multiplied to find the amount we needed to add to the tubes to get this concentration.

(100uM)x = (5uM)(20uL)
x = 1 uL

We added 1 uL of each oligo to the tubes.

We had six total tRNAs we needed to assemble so each of us took one FML (one person took two) and to each tube we added 1uL of each oligo for a total of 7uL, 2uL of buffer, and 11uL of water for a total volume of 20 uL. Five of the oligos were constant and 2 varied for each of the different tRNAs. So everyone pipetted the same 5 oligos and everyone also had 2 unique oligos to add. When all of the oligos were added and the tubes ready, we boiled the tubes at 50C for 5 minutes. We then allowed the tubes to remain in the water to cool very slowly overnight so that the annealing process could take place.

The last thing we did at the end of the day was finish off our plasmid digestion that we had begun in the morning. We added the buffer, DNA, water, and restriction enzymes and placed this in the incubator for overnight digestion.

'''To Do''':

1. Isolate our desired dsDNA from the PCR.
2. Verify our tRNA inserts and eventually transform these into plasmids.
3. Check our agar plates to see colonies that have replicated our pLac+RBS, pBAD+RBS, and pBAD genes.

==Friday, June 12, 2009==

This morning, we began by outlining the main items on today's agenda.

1. tRNAs: Ligation, Transformation
2. Digestion reporters (run all of it and gel purify)
3. Transformed cells
4. PCR: Gel run an aliquot to verify (5uL)

We used the equation given by the Ligation protocol to calculate how many nanograms of insert we would need. Initially, we started out calculated the MW average of the 9 and 10 anticodon loops of tRNAs which were 15828.23 ug/umols and 159016.6 ug/umols respectively. We then saw, however, that these would not be helpful values in the process. Instead we started out with the equation below to find the final ng of insert we would need for the ligation.

[[Image: ligationcalculations.png]]

We calculated our initial concentration in ng/uL of insert as shown below.

[[Image: ligationequation.png]]
X nanograms of insert = [(2)(144bp)(50 ng linearalized plasmid)]/2038bp
X = 7.0657 ng of insert

We wanted to find the amount of uL of insert we would need to use. We diluted the initial concentration 50x because we wanted a pipettable amount of insert. Since we knew we wanted the final amount of nanograms to be 7.0657, we calculated as shown below:

''DILUTION'': 555.48/50 = 11.012
''CALCULATION'': 11.012 ng/ 1uL = 7.065 ng/ x uL
x = .64 uL of insert

After we had done these calculations, half of the group started to carryout the ligation of the tRNA inserts into empty plasmids. We needed 7 ligations total: 6 were the tRNAs we needed, while the last one is a negative control. These are the final amounts of what we used:

[[Image: Trnaligation.png]]

The other half of the group (which included me) prepared the agarose gels so that we could run gels for the digested reporters and the PCR inserts. We calculated that the digested reporters needed an agarose of 4% and the PCR reporters needed an agarose of 1.45%. We allowed this gel to form for about 45 minutes to an hour. We prepared both the gels and allowed them to run through lunch.
After lunch, we took pictures of our gels and noticed that the third lane in the PCR gel (third lane from the top in the bottom photo) did not develop a band. The lane was supposed to contain the CUAGU insert for Tetracycline Resistance.

[[Image: 2009-06-12 15hr 54min.jpg]] [[Image:2009-06-12_16hr_07min.jpg]]

Romina Clemente and I decided to repeat this PCR using the TetResistance and RBS+TetResistance plasmids as templates. We had to recalculate how much template DNA to use. We let the PCR run overnight. We also verified that the digestion fragments (both the plasmid and the insert) on the other gel were the correct size. The plasmids, which are the brighter fragment in the gel with only 2 lanes, contain the plasmid base pairs along with the rest of our reporter genes. We isolated these plasmids from the gel today and stored them for purification on Monday.

In the meantime, other members of our lab had finished the ligation of the tRNA genes to the empty plasmids and were now transforming these into competent cells. They placed the agar plate with these cells in the fridge overnight.

We also observed the transformations that we had performed yesterday and noticed that I13453 did not form any colonies. J04500 and J3102 did form colonies.

To Do:
1. PCR: Dig, Ligate, Transform
Clean and concentration column, digest, clean and concentration column, ligate.
2. Gel purification of our digestion plasmids
3. Check if colonies have grown for our tRNAs

IGEM Notebook

2009-06-12T23:02:01Z

ShPunjabi: /* Friday, June 12, 2009 */

==Wednesday, June 3, 2009==

Romina Clemente and I are trying to find suitable reporter proteins to use. Yesterday, Leland Taylor and Alyndria Thompson were working on ways to insert the gene sequences into the plasmid. Upon seeing how they wanted to manipulated the reporter gene to include the logical clauses, we came up with a few criteria for the reporter genes we would use. The following criteria for genes are listed in order of the broadest aspect to look at to the narrowest aspect:
a) Doesn't contain restriction sites for the 4 restriction enzymes (EcoR1, Xbal, Spel, Pst1) used to cleave the Biobrick part out of the plasmid.
b) Contains 6 cutter restriction sites.
c) These restriction enzymes aren't blunt (cleave straight down at one spot).
d) These restriction sites are close to thge 5' (beginning) end of the sequence.
e) These enzymes are easiest to work with and cheapest.
We are finding the part numbers of the reporter genes we want to use (antibiotic resistance, fluorescence, LacZ) through our own GCAT because we know these ones work. We are then locating these parts on the parts registry [1] website. We copied and pasted the gene sequences we obtained from the registry onto the ApE software [2]. From here, we were able to generate a genetic map of each gene that outlined each restriction site that fit our criteria. We put each genetic map, alongside the part number used, into a Word document.

http://gcat.davidson.edu/GcatWiki/images/0/0e/Restriction_Site_Mapping_on_Reporter_Genes.doc

Later on in the day, we decided that the best way to test for suppression would involve placing the 5mers at the beginning of the reporter gene instead of inserting them into the reporter gene. To do this, we decided that the gene along with its start codon need to be expressed after the 5mer. We will insert a BioBrick plasmid with a 5mer and start codon that will be incorporated before the gene:
RBS-6-8nt-ATG-5mer--ATG-gene

Leland Taylor assembled the oligos that we will need for the BioBrick pieces.

In order to test whether the reporter protein would be expressed regardless of the 5mer being suppressed or not, we would need to remove the suppressor tRNAs from the cell.

Meanwhile, I helped figure out the coding sequence that we will need for the suppressor tRNAs that will bind to the 5mers. We decided that we would be using the following suppressor tRNAs on the Davidson side: CUAGU, CCCUC, CGGUC, CCAUC, and CCACU. We searched through several papers, with little result and finally emailed Dr. Christopher Anderson of UC Berkeley to request the DNA sequence. He emailed us back with a generic DNA code that we could use to create the tRNA; however, for each suppressor tRNA we would use, we would need to change base pairs in the anticodon loop.

The length of the tRNA is 92 nt. The anticodon loop is comprised of 9 nt. Once we have BioBricked the DNA sequence with 4 restriction sites and supplemental nucleotides, the length of the entire gene is 144 nucleotides. This is made up of the 92 nt +22 nt on each side of the sequence (44) + 4 nt on each side of the restriction ends (8).

At the end of the day, we decided to use tetracycline resistance and RFP as our reporter proteins for this tester experiment.

==Thursday, June 4, 2009==

This morning, I began the day by learning how to use the PEARL prgram. Olivia Ho-Shing sat with me and walked me through the program script. And then, Leland Taylor and Shashank Suresh had a problem they wanted us to solve. Leland explained that we need to make sure there is a stop codon in the gene if we fail to suppress the 5mer. The stop codon needs to be before we find another 5mer. They also wanted us to run through both the RFP and Tet Resistance genes to make sure that they did not contain any of the anticodon sequences from our 5 frameshift suppressor tRNAs so as to throw the translation out of frame. They further wanted us to confirm that if we do have successful suppression, there are no stop codons in the middle of the RFP and Tet Resistance genes. PEARL did not find any matches for the suppressor tRNAs within genes. We found 3 matches for stop codons UAA. Two of these occured at the end of the Tet and one at the end of RFP, where they should normally be to end translation into a protein.

Following this, Olivia Ho-Shing and I tried to determine how we can add BioBrick ends to our single stranded DNA sequences that code for the suppressor tRNAs. Our ultimate goal is to find complements for these single strands so they can be put into plasmids. We found the standard prefixes and suffixes for sequences that do not contain ATG (becuase this sequence is a functional RNA and will not be translated) and we placed these before and after the altered 92bp sequence that Dr. Anderson provided us with. The prefixes and suffixes are necessary because they are the extra nucleotides that will be mimicking a "restriction enzyme cutting site". The single strand appeared as below after the Bio Brick prefixes and suffixes were added:

22ntPREFIX--32nt--VARIABLE ANTICODON LOOP--51nt--21ntSUFFIX

We used the suppressor codon CUAGU as our example and placed its appropriate anticodon loop in the sequence. We put this into the lancelator that would give us the other strand as well as the oligos we would need for ideal construction of this double stranded DNA fragment to put into the plasmid. We recieved a total of 4 oligos, 2 of which were variable and 2 of which were constant for all 6 different suppressor tRNAs. Using this double strand, we simulated a restriction enzyme digestion by EcoRI and PstI. We took off the G at both ends of the top strand and then removed the first five nucleotides on each end of the bottom strand. This made the top strand longer than the bottom one. We then proceeded to take the two variable strands and changed the anticodon loop nt for each suppressor tRNA we used.

Around lunchtime, we encountered a problem with the BioBrick scar in the "ATG-5mer subpiece" of the test project. It turns out that after annealing the sticky ends of the Xba1 and Spe1 sites, the scar created had a stop codon TAG (UAG) that was in frame with the suppressed codon. When the Xba1 site on insert anneals to the Spe1 site on the plasmid (the green portions), we get a scar that reads TACTAG.
[[Image:screen-capture.png]]

For the afternoon, Olivia Ho-Shing and I finished assembly of the oligos of the suppressor tRNA codes. We assembled the document below:

http://gcat.davidson.edu/GcatWiki/images/5/5e/TRNAoligostoOrder.doc

We were assigned tasks of creating "cartoons" to illustrate the phenomenon that had occurred. No one could successfully do this however, because we could not understand how the piece had formed. The parts registry website provided wrong information on the restriction site of Spe1 which slowed our efficiency. This project turned into an effort to try to find two other different restriction enzymes that have complementary sticky ends other than Xba1 and Spe1. We want to get rid oc the scar that contains TACTAG.

We ended up deciding that a hybrid of two ideas was needed: restriction enzymes would need to be used to make several reporter gene sequences with different beginnings dependending on the suppressor tRNA sequence needed.

==Friday, June 5, 2009==

Our goal for this morning was to discuss the hybrid PCR approach to assemble the part that comes before the gene we wanted to express. We decided that we would have a BioBrick prefix and a 5mer followed by a sequence of nucleotides whose identity was up to us. Using PCR we would get the second double strand if we have the following:

BioBrick Prefix--ATG--5mer--gene until we get to restriction site

The PCR primer would attach to the ATG and the 5mer and would replicate the complementary base pairs to make a double stranded DNA sequence. As several of us were confused about PCR methods, we looked at the online link provided by Alyndria Thompson's link to the PCR interactive website which was very helpful. We also did some research to discover that the restriction enzyme BseRI (which is contained in the YFP that we wanted to alter) is not compatible with any other restriction enzyme. This is good because we did not want to plasmid containing this gene to close up with Xba1 once we digested it.

The team experienced some confusion with the hybrid idea and spoke to Clif Davis of Missouri Western University on the phone. He told us that there will be one part that is a fragment of the beginning of some nucleotides as a restriction cleaving site, ATG, the logical clause, and the start of the YFP gene that will be placed into a plasmid containing a space. To the left of the space will be the BioBrick Prefix and the right of the space will contain the rest of the genes in the YFP. Therefore, we decided not to alter any BioBrick ends and just look at restriction sites at the beginning of reporter proteins. The small fragment that will be placed into the plasmid will be made by PCR. The PCR will attach a primer onto the ATG and 5mer and replicate.

While we were discussing this, we expanded upon the idea that we can do whatever we want with the "suffix" portion between the 5mer and the beginning of YFP. We decided it would be best to remove ATG and add the first 5 amino acids and the restriction enzyme site. Along with this, if we have the 5mer coding for a certain amino acid (ie methionine) we could replace another codon that codes for methionine WITH that 5mer so long as this codon is contained within the first 15 nt. This way, we would not be altering the protein.

Olivia Ho-Shing came up with an idea of cascading PCR effect for longer logical clauses. Post translational modification of the protein was another one of her ideas. This applies because although we may code for the protein in a given way, the protein may modify or fix itself up to perform a slightly different function in its tertiary form.

Dr. Campbell suggested that we can look up restriction enzymes 400-500 base pairs into the reporter protein we would like to use; however, we have more flexibility in our procedure if we choose more "hardy" cutters. So instead, we could cut with EcoRI instead of with Xba1.

During the afternoon, we set about three tasks:
1) Which primers to use
2) Which promoters to use
3) Which restriction enzymes and how they will determine use of reporter proteins

Olivia Ho-Shing and I began looking up which restriction enzymes were strong so we could base our reporters on that. We began with Tet and RFP since those were the two reporters we had started out with choosing. I found a website that provided enzyme cleavage efficiency based upon the proximity of the site to the end of the PCR fragment that contains it. We discovered the following based upon the attached documents:

http://gcat.davidson.edu/GcatWiki/images/8/8f/Restriction_Enzyme_Cleavage_Efficiency.doc
http://gcat.davidson.edu/GcatWiki/images/9/9a/07Jun09_RE_Cutting_Efficiency_oh.doc

We decided that these two reporters contained efficient enzymes in a good quantity to choose from. On Monday, we will look through these enzymes once more and finalize which enzymes we would like to use with RFP and Tet.

==Sunday, June 7, 2009==

We did preparations for the mini prep this evening. The following notes are courtesy of Olivia Ho-Shing.

1. Sterically transfer ~2mL LB+AMP (AMP are ampicillin resistant plasmids) *Never put fluid back into bottle once it has entered pipette*
2. Use forceps to grab toothpick, scrap some frozen cells, rub in tube with top

'''Tubes'''

S03511 (2)

K091111

B0030

I715039

K091112

I715039

J31007 (2)

B0034

E1010

S03710

==Monday, June 8, 2009==

We reviewed the PCR process this morning as well as the restriction enzyme cleavage process.

Then, Leland Taylor, Olivia Ho-Shing and I all worked on converting the tRNA oligos we made to the template that Missouri Western used. The oligos we had previously made were different from the ones that Dr. Anderson had originally emailed us because further communication between Dr. Anderson and Missouri Western resulted in a better template.

We tried several times to minimize the number of oligos while keeping their individual lengths under 80bp. We managed to do this in the end by creating 7 oligos, 2 of which are variable between the suppressor tRNAs.
We reviewed the following document and finalized the oligos that we needed to order for the frameshift suppressor tRNAs. We decided to keep both the 10bp and 9bp anticodons of CCAUC.

I also looked up the amino acids that corresponded to the FS (frameshift suppressor) tRNAs for future use:

1. CUAGU = Leu
2. CCCUC = Pro
3. CGGUC = Arg
4. CCACU = Pro
5. CCAUC* = Pro
*9 and 10 bp anticodons

Also, we have decided to use NcoI as the restriction enzyme for RFP. This cleavage site occurs 419 bp into the gene. We have decided to use BamHI as the enzyme for Tetracycline Resistance and the cleavage site occurs 290bp into the gene. Both of these enzymes are very powerful and hardy.

We also looked over Missouri Western's tRNA oligos and primer oligos which we approved.
Our oligos were also approved by Missouri Western and Dr. Campbell ordered them while we supervised.

In the afternoon we did the miniprep. The mini prep directions were as appear in the link below.

http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_MiniPrep.html

We then worked with the Nano Prep to dilute our solution of DNA to an absorption of 1.5 and then we made labels for the tubes. The chart below shows relevant information we obtained from the NanoDrop for each of the reporter plasmids.

[[Image: Screen-capture-6.png]]

==Tuesday, June 9, 2009==

'''Preparations for restriction enzymes digestion of plasmids'''

Purpose: To determine the size of the plasmid inserts and ensure that plasmids are what we extracted from the cells during the mini-prep.

Things we need:
DNA ~3uL
Buffer 10x (Want to dilute to 1x) so add 2uL
Enzyme 1 ~1uL
Enzme 2 ~1uL
Water ~13uL
Final Volunter about 20 uL.

Need to choose the right buffer for the restriction enzymes. All buffers come in 10x (10 times more concentrated than what you want it to be when you use it). [http://gcat.davidson.edu/GcatWiki/index.php/Davidson_Missouri_W/Double_Digest_Guide Buffer Website]

Total volume of enzymes has to be 10% or less than final volume (most volume 2uL). Barely submerge the pipet tip into the solution when pipeting the enzymes.
Star (*) activity: Restriction enzyme stops cutting specifically and starts cutting everywhere.

DNA can't be seen by itself so we add EtBr (the bigger the insert, the more will bind) so bigger strands appear brighter. samples.

The order of entry doesn't matter, but make sure to enter the enzymes last.

Will have lots of labeled tubes and will put 3uL of appropriate plasmids into the tubes.
In the meantime, we'll make a "Master Mix" of all the other ingredients in a bigger tube. Always make enough volume for one extra tube.

Enzymes usually have an optimal temperature of 37C. We will let this reaction go for an hour and then we will run it on a gel.

'''Gel Electrophoresis Protocol'''

Website online for ideal gel concentration for resolving different sizes of molecules [http://gcat.davidson.edu/iGEM08/gelwebsite/gelwebsite.html Gel Concentration Website].
See protocol on the Davidson/Missouri Western Wookie for running molecules in the gel and how to digest with restriction enzymes.

'''What's Next?'''

1. 5' end additions
a. Using PCR
b. Cut at the restriction sites EcoR1 and either Bam H1 or Nco1
c. Cut plasmid and reporter with same restriction enzymes as above
d. Run a gel and purify "keeper" DNA (Ligate transform, screen, sequence)
e. Then, we take the "keeper" DNA and combine it with the insert from part

We can have steps d and e ready in advance.

2. tRNAs
a. Assemble the oligos into genes (Requires lots of calculations beforehand, but the actual steps )
b. Plasmids cut up with EcoR1 and Pst1 '''WE CAN USE THE "WASTE" PLASMID FROM THE RESTRICTION ENZYME DIGEST'''
c. Gel purify the plasmids (Ligate transform, screen, sequence)

The following tables include Master Mix measurements, agarose gel concentrations and buffers.
http://gcat.davidson.edu/GcatWiki/images/0/08/Shamitapic3.png
http://gcat.davidson.edu/GcatWiki/images/d/d1/Shamitapic4.png
http://gcat.davidson.edu/GcatWiki/images/6/6f/Shamitapic5.png

We made the Master Mix with our DNA and put it in the incubator at 10:40 AM.

'''Preparing the Gels'''

Now, we will be making 2 gels, 1 at 0.8% and 2.5% (weight to volume percent). The gel volume is 60mL.

'''Definition of 1% solution:
1 gram of agarose/ 100mL buffer'''

So for a 0.8% solution: 0.8 grams agarose/100mL buffer AND we need 60mL (fixed volume for casts run) so multiply that by 0.6.
Final concentration:

.48 grams agarose/60mL buffer

1 uL of EtBr stock for 60mL of gel.
EtBr is in the buffer so it is constantly replenishing itself even though it is moving toward one end in order to make bands on the other end visible.

After Dr. Campbell's demonstration, we each helped prepare a 0.8% buffer solution using the agarose powder, buffer and EtBr. We mixed together the first two substances in a 200mL Erlenmeyer flask and heated it for 120 seconds in the microwave. We then added EtBr and poured the gel solution into the gel cast.

'''Agar Plate'''

After lunch, we grew some more cells for each of the 11 parts in an agar plate labeled "LBAMP-AMC-9 June" so that so would have extra cells that could be stored for up to 2 weeks.
We made small circles on the agar plate and numbered them 1-11. The numbers corresponded to the following parts. We put the plate in the incubator overnight to let the cells grow.

1. B0030 AMC
2. K091111
3. S03710
4. I715039 OEH
5. S03511 ORH
6. I715039 AST
7. B0030 SDP
8. K091112
9. J31007 LJH
10. J31007 REC
11. S03511

'''Unloading the Gels'''

The following shows which parts we entered in which gels:

[[Image: screen-capture-2.png]]

We unloaded the 2.5% agarose first and after observing the bands under a UV light, put the gel into the BioRad machine to observe the bands. The image below is of a Molecular Weight Marker that shows the number of bp in control fragments of DNA according to the distance they travel down the gel.

[[Image: screen-capture-1.png]]

We obtained the following printout for the first gel that was 2.5% Agarose.
[[Image: 2009-06-09_14hr_44min.jpg]]

The bands in the first well at the bottom are those of the molecular weight marker. We skipped the lane above (to the right) of the MWM and entered the rest of the inserts as described in the above table. A light band is visible for each of the 4 other wells. In Lanes 3 and 4 we concluded we are supposed to have approximately 45 bp because the insert is 15bp + 30 bp on either side of the insert (Xba1 and Spe1 restrictions sites + sticky ends + extra bases). We used the same logic for Lanes 5 and 6 which are supposed to contain approximately 86 base pairs.

One discrepancy in this gel, however, is that Lanes 5 and 6, which according to their labels respectively contain the LacIQ promoter and the Lac IQ1 promoter. According to the information online at the parts registry website, pLacIQ1 should be the same length as pLacIQ. The gels do not show this, but instead show that the sample in Lane 5 is smaller than that in Lane 6 because Lane 5 traveled further. Leland Taylor and Romina Clemente sent out an email to Pallavi Penumetcha to ask about this because she had written a paper about this.

We then unloaded the 0.8% agarose gel and then placed this in the BioRad machine under the same protocol as above. The following image is the 0.8% Agarose.

[[Image:2009-06-09_15hr_21min.jpg]]

We observed that the bands in Lane 1 (which is now at the top) are examples of high intensity bands. The plasmids contained in the original well are twice the intensity of the insert.

In Lane 5, however, we observed several different bands. This could be due to the extra DNA that was sitting on the top of the well. We decided to re-expose the gel to get better observations on Lanes 3 and 4 because the bands were not as intense. We anticipated seeing a band at about 250 bp because this was the size of the insert (plus extra bp). After re-exposing the gel, we did not find any difference and still did not see any bands in these lanes. We have decided to redo the gel on the part S03511 and so we prepared the master mix with that DNA and incubated it so that we could run the gel tomorrow.

We also cut out the "empty" plasmids from the gel so that we could use this as vectors for the coding sequence of the suppressor tRNAs.

Tomorrow we will be planning out specific tasks for people once the oligos arrive.

==Wednesday, June 10, 2009==

We began this morning by making the agarose gel with the same protocol as yesterday. This time, since we are just re-doing both S03511 inserts, we will have a 1.8% agarose gel.

'''OUTLINE: WHERE DO WE STAND?'''

Yesterday
a. Isolated empty plasmids
b. Verified most inserts
c. Waiting for:
>tRNA oligos
>Primers
a. Will amplify the reporter gene in the plasmid itself

Next?
a. When Primers Arrive
> Amplify the reporter genes with primers (PCR takes ~3 hours)
> Calculate how much reporter plasmid we will need (Start with 50x more than we actually will need)
> Cut amplified sequence (EcoR1 and BamH1/Nco1) then purify what we need
> Cut reporter plasmid (EcoR1 and BamH1/Nco1) then purify what we need
> Ligate amplified sequence and reporter plasmid
b. When tRNA oligos arrive
> Calculate how much of each oligo we will need (takes a while...)
> "Boil and cool" tRNA oligos together
> Ligate fragments (dsDNA) into plasmids we isolated yesterday (~5 minutes)
> Transform the plasmids (~20 minutes)

* LOOK THROUGH LIGATION PROTOCOL (How to assemble genes)
* LOOK THROUGH TRANSFORMATION PROTOCOL

''What we need to get done today'':
1. Prepare the control for the Tet Resistance by BioBricking the part pLac+RBS (S03511) with the TetA (J31007).
2. Check the part number for RFP and also for pLac+RBS+RFP
3. Read through ligation protocol
4. See how many nanograms of plasmid we will need for the tRNAs and for the inserts and multiply this number by 50.
> Check if we have enough plasmids from our mini-prep
5. Purify the plasmids from the gel

After we made this outline, Leland, Olivia and I analyzed the gel we ran this morning and did not see a more than one band in the 2 lanes that contained DNA from S03511 (image below). Since we did not see multiple bands in yesterday's gel either, we conclude that there is something wrong with the DNA rather than with the restriction enzymes that we used to cut it. We concluded that there is probably not an insert in this plasmid both the part and the plasmid are useless.

[[Image: 2009-06-10 10hr 30min.jpg]]

Now, we do not have an immediately accessible promoter. We have decided to order the pBAD promoter from Missouri Western and we will attach the RBS+Tet to this plasmid. Our insert for this will be the larger gene because it is easier to manipulate in the gel than the small gene.

Alyndria Thompson and Romina Clemente are figuring out the size of the plasmids including the reporter genes.

After lunch we performed several steps to isolate and purify the gel to get the empty plasmids will be the vectors for the tRNA DNA. Then, we used the NanoDrop to assess the purity of our DNA. We found that the plasmids were more pure than we had expected. The following values were obtained:

Abs 1.850
A-260 10 mm path 0.172
A-280 10 mm path 0.075
260/280 2.28
260/230 0.09

We then followed the protocol for preparing the agar plates. We put the plate with the agar solution in the Autoclave.

While the solution was in the autoclave, we discussed that we don't really need a promoter in front of RBS+Tet for the gene to be expressed because Tet expression is so strong that the gene would translate without a promoter. However, it would be a good idea to have the promoter because if it was possible to reinforce the translation of the reporter protein, we wanted to do that.

We found that there were some tubes in the lab fridge that contained the pBAD and pLac promoters with RBS. We decided to use these as the promoter and RBS for our experiments. The pLac promoter will be used for RFP and the pBad promoter used with Tet Resistance. We decided to use the pBad with Tet because it is weaker in comparison to pLac. As expression of the Tet gene can be toxic to a cell, we do not want to overexpress the gene with a stronger promoter.

Once the agar solution was sterilized in the Autoclave, we removed it, cooled it, and pipetted the antibiotic resistance into it. We then poured the solution into the plates, removed any bubbles, and let the gel form.

Alyndria Thompson and Romina Clemente concluded that we had more than enough recieving vectors for both the RFP and Tetracycline genes. Since we need 50 nanograms, it is wise to have at least 50 times that much that we can work with, so we need at least 2,500 ng.

RFP 1: 77.6 x 4 = 310.4 ng/uL
x 20 uL = 3,272 ng
RFP 2: 59.5 x 4 = 238 ng/uL
x 20 uL = 4,760 ng
Tet: 40.9 x 4 = 163.6 ng/uL
x 20 uL = 3,272 ng

According to these calculations we have more than 50 times what we need for both instances of RFP and Tet.

Tomorrow, we can prepare the Master Mix for the digestions of both reporter plamids and then add the restriction enzymes once they arrive. Then, we can follow through with the rest of the plans we had made above in the section titles "Next?".

==Thursday, June 11, 2009==

We began this morning by calculating how many uL we would need for each of the mixes for digesting our reporter plasmids. We decided to double the final volume of each mix to 40uL instead of 20uL. Since we knew the concentration of each plasmid and also knew we needed 2500 ng, we cross multiplied to find the uL of DNA we would need for each.

Concentration Calculations:
310.4 ng/1 uL = 2500ng/x uL
x = 8.05 uL

Here is a chart indicating how much of each we used for the 2 mixes.

[[Image: DoubledigestRFPTetR.png]]

On the Promega website, we found a list of compatible buffers for double digestions. Buffers E and H are compatible for the Tet plasmid and RFP plasmid digestions respectively. We fortunately have both buffers in stock.

[[Image:BamEcoR1.png]] [[Image:NcoEcoR1.png]]

We waited for our waited for the restriction enzymes to arrive and in the meantime, we transformed the plasmids with pLac+RBS, pBad, and pBad+RBS into ''E.coli'' cells. We want the ''E.coli'' cells to replicate the plasmids with these genes because we have a limited amount of genes. [http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_Transformation.html Zippy Plasmid Transformation]

Once our oligos and restriction enzymes arrived, we got to work right away!

'''PCR (Polymerase Chain Reaction)'''

We calculated how much plasmid we will need for the PCR. We decided to dilute the concentration of our plasmids a hundred fold so that we could get pipetable amounts.

''Change in Concentrations''
310.4 ng/uL x 1/100 (dilution factor) = 3.104 ng/uL (RFP)
163.6 ng/uL x 1/100 (dilution factor) = 0.164 ng/uL (Tet)

''Example shown with RFP''
3.104 ng/1 uL = 1ng/ x uL, x = .32
For Tet, x = .61

We also calculated how much of each primer we would need with the following equation:

C1V1 = C2V2
(100 uM) (x) = (1 uM)( 100 uL)
x = 1 uL

[[Image: screen-capture-8.png]]

Each of the 5 of us made had 2 tubes (one for each reporter protein) because there were 5 different forward primers.To each tube, we added the template DNA (either RFP or Tet), water, the respective reverse primer (either RFP or Tet), and the forward primer depending on which 5mer we had chosen. My 5mer was CCAUC. Finally, we added the Master Mix last because this contained the enzyme (DNA polymerase) that would amplify our desired sequence.

[[Image: Pcrtubes.png]]

We followed the temperature cycles so that PCR could occur. The annealing temperature needed to be 5C less than the smallest melting temperature of the primers. Since the reverse primer of RFP had a melting temp of 66C, we set the temperature of the 3rd cycle on 61C.

[[Image: Temperaturecycle.png]]

'''tRNA Oligo Assembly'''

We needed to calculate the concentration of each of the oligo before carrying out the assembly. The stock concentration of each of the oligo was 100 uM. Since we wanted to have 5uM of the oligo in the test tube, and we know that final volume of the test tube is 20 uL, we just cross multiplied to find the amount we needed to add to the tubes to get this concentration.

(100uM)x = (5uM)(20uL)
x = 1 uL

We added 1 uL of each oligo to the tubes.

We had six total tRNAs we needed to assemble so each of us took one FML (one person took two) and to each tube we added 1uL of each oligo for a total of 7uL, 2uL of buffer, and 11uL of water for a total volume of 20 uL. Five of the oligos were constant and 2 varied for each of the different tRNAs. So everyone pipetted the same 5 oligos and everyone also had 2 unique oligos to add. When all of the oligos were added and the tubes ready, we boiled the tubes at 50C for 5 minutes. We then allowed the tubes to remain in the water to cool very slowly overnight so that the annealing process could take place.

The last thing we did at the end of the day was finish off our plasmid digestion that we had begun in the morning. We added the buffer, DNA, water, and restriction enzymes and placed this in the incubator for overnight digestion.

'''To Do''':

1. Isolate our desired dsDNA from the PCR.
2. Verify our tRNA inserts and eventually transform these into plasmids.
3. Check our agar plates to see colonies that have replicated our pLac+RBS, pBAD+RBS, and pBAD genes.

==Friday, June 12, 2009==

This morning, we began by outlining the main items on today's agenda.

1. tRNAs: Ligation, Transformation
2. Digestion reporters (run all of it and gel purify)
3. Transformed cells
4. PCR: Gel run an aliquot to verify (5uL)

We used the equation given by the Ligation protocol to calculate how many nanograms of insert we would need. Initially, we started out calculated the MW average of the 9 and 10 anticodon loops of tRNAs which were 15828.23 ug/umols and 159016.6 ug/umols respectively. We then saw, however, that these would not be helpful values in the process. Instead we started out with the equation below to find the final ng of insert we would need for the ligation.

[[Image: ligationcalculations.png]]

We calculated our initial concentration in ng/uL of insert as shown below.

[[Image: ligationequation.png]]
X nanograms of insert = [(2)(144bp)(50 ng linearalized plasmid)]/2038bp
X = 7.0657 ng of insert

We wanted to find the amount of uL of insert we would need to use. We diluted the initial concentration 50x because we wanted a pipettable amount of insert. Since we knew we wanted the final amount of nanograms to be 7.0657, we calculated as shown below:

''DILUTION'': 555.48/50 = 11.012
''CALCULATION'': 11.012 ng/ 1uL = 7.065 ng/ x uL
x = .64 uL of insert

After we had done these calculations, half of the group started to carryout the ligation of the tRNA inserts into empty plasmids. We needed 7 ligations total: 6 were the tRNAs we needed, while the last one is a negative control. These are the final amounts of what we used:

[[Image: Trnaligation.png]]

The other half of the group (which included me) prepared the agarose gels so that we could run gels for the digested reporters and the PCR inserts. We calculated that the digested reporters needed an agarose of 4% and the PCR reporters needed an agarose of 1.45%. We allowed this gel to form for about 45 minutes to an hour. We prepared both the gels and allowed them to run through lunch.
After lunch, we took pictures of our gels and noticed that the third lane in the PCR gel (third lane from the top in the bottom photo) did not develop a band. The lane was supposed to contain the CUAGU insert for Tetracycline Resistance.

[[Image: 2009-06-12 15hr 54min.jpg]] [[Image:2009-06-12_16hr_07min.jpg]]

Romina Clemente and I decided to repeat this PCR using the TetResistance and RBS+TetResistance plasmids as templates. We let the PCR run overnight. We also verified that the digestion fragments (both the plasmid and the insert) on the other gel were the correct size. The plasmids, which are the brighter fragment in the gel with only 2 lanes, contain the plasmid base pairs along with the rest of our reporter genes. We isolated these plasmids from the gel today and stored them for purification on Monday.

In the meantime, other members of our lab had finished the ligation of the tRNA genes to the empty plasmids and were now transforming these into competent cells. They placed the agar plate with these cells in the fridge overnight.

We also observed the transformations that we had performed yesterday and noticed that I13453 did not form any colonies. J04500 and J3102 did form colonies.

To Do:
1. PCR: Dig, Ligate, Transform
Clean and concentration column, digest, clean and concentration column, ligate.
2. Gel purification of our digestion plasmids
3. Check if colonies have grown for our tRNAs

File:Ligationequation.png

2009-06-12T22:55:14Z

ShPunjabi:

File:Ligationcalculations.png

2009-06-12T22:54:51Z

ShPunjabi:

IGEM Notebook

2009-06-12T22:53:29Z

ShPunjabi: /* Friday, June 12, 2009 */

==Wednesday, June 3, 2009==

Romina Clemente and I are trying to find suitable reporter proteins to use. Yesterday, Leland Taylor and Alyndria Thompson were working on ways to insert the gene sequences into the plasmid. Upon seeing how they wanted to manipulated the reporter gene to include the logical clauses, we came up with a few criteria for the reporter genes we would use. The following criteria for genes are listed in order of the broadest aspect to look at to the narrowest aspect:
a) Doesn't contain restriction sites for the 4 restriction enzymes (EcoR1, Xbal, Spel, Pst1) used to cleave the Biobrick part out of the plasmid.
b) Contains 6 cutter restriction sites.
c) These restriction enzymes aren't blunt (cleave straight down at one spot).
d) These restriction sites are close to thge 5' (beginning) end of the sequence.
e) These enzymes are easiest to work with and cheapest.
We are finding the part numbers of the reporter genes we want to use (antibiotic resistance, fluorescence, LacZ) through our own GCAT because we know these ones work. We are then locating these parts on the parts registry [1] website. We copied and pasted the gene sequences we obtained from the registry onto the ApE software [2]. From here, we were able to generate a genetic map of each gene that outlined each restriction site that fit our criteria. We put each genetic map, alongside the part number used, into a Word document.

http://gcat.davidson.edu/GcatWiki/images/0/0e/Restriction_Site_Mapping_on_Reporter_Genes.doc

Later on in the day, we decided that the best way to test for suppression would involve placing the 5mers at the beginning of the reporter gene instead of inserting them into the reporter gene. To do this, we decided that the gene along with its start codon need to be expressed after the 5mer. We will insert a BioBrick plasmid with a 5mer and start codon that will be incorporated before the gene:
RBS-6-8nt-ATG-5mer--ATG-gene

Leland Taylor assembled the oligos that we will need for the BioBrick pieces.

In order to test whether the reporter protein would be expressed regardless of the 5mer being suppressed or not, we would need to remove the suppressor tRNAs from the cell.

Meanwhile, I helped figure out the coding sequence that we will need for the suppressor tRNAs that will bind to the 5mers. We decided that we would be using the following suppressor tRNAs on the Davidson side: CUAGU, CCCUC, CGGUC, CCAUC, and CCACU. We searched through several papers, with little result and finally emailed Dr. Christopher Anderson of UC Berkeley to request the DNA sequence. He emailed us back with a generic DNA code that we could use to create the tRNA; however, for each suppressor tRNA we would use, we would need to change base pairs in the anticodon loop.

The length of the tRNA is 92 nt. The anticodon loop is comprised of 9 nt. Once we have BioBricked the DNA sequence with 4 restriction sites and supplemental nucleotides, the length of the entire gene is 144 nucleotides. This is made up of the 92 nt +22 nt on each side of the sequence (44) + 4 nt on each side of the restriction ends (8).

At the end of the day, we decided to use tetracycline resistance and RFP as our reporter proteins for this tester experiment.

==Thursday, June 4, 2009==

This morning, I began the day by learning how to use the PEARL prgram. Olivia Ho-Shing sat with me and walked me through the program script. And then, Leland Taylor and Shashank Suresh had a problem they wanted us to solve. Leland explained that we need to make sure there is a stop codon in the gene if we fail to suppress the 5mer. The stop codon needs to be before we find another 5mer. They also wanted us to run through both the RFP and Tet Resistance genes to make sure that they did not contain any of the anticodon sequences from our 5 frameshift suppressor tRNAs so as to throw the translation out of frame. They further wanted us to confirm that if we do have successful suppression, there are no stop codons in the middle of the RFP and Tet Resistance genes. PEARL did not find any matches for the suppressor tRNAs within genes. We found 3 matches for stop codons UAA. Two of these occured at the end of the Tet and one at the end of RFP, where they should normally be to end translation into a protein.

Following this, Olivia Ho-Shing and I tried to determine how we can add BioBrick ends to our single stranded DNA sequences that code for the suppressor tRNAs. Our ultimate goal is to find complements for these single strands so they can be put into plasmids. We found the standard prefixes and suffixes for sequences that do not contain ATG (becuase this sequence is a functional RNA and will not be translated) and we placed these before and after the altered 92bp sequence that Dr. Anderson provided us with. The prefixes and suffixes are necessary because they are the extra nucleotides that will be mimicking a "restriction enzyme cutting site". The single strand appeared as below after the Bio Brick prefixes and suffixes were added:

22ntPREFIX--32nt--VARIABLE ANTICODON LOOP--51nt--21ntSUFFIX

We used the suppressor codon CUAGU as our example and placed its appropriate anticodon loop in the sequence. We put this into the lancelator that would give us the other strand as well as the oligos we would need for ideal construction of this double stranded DNA fragment to put into the plasmid. We recieved a total of 4 oligos, 2 of which were variable and 2 of which were constant for all 6 different suppressor tRNAs. Using this double strand, we simulated a restriction enzyme digestion by EcoRI and PstI. We took off the G at both ends of the top strand and then removed the first five nucleotides on each end of the bottom strand. This made the top strand longer than the bottom one. We then proceeded to take the two variable strands and changed the anticodon loop nt for each suppressor tRNA we used.

Around lunchtime, we encountered a problem with the BioBrick scar in the "ATG-5mer subpiece" of the test project. It turns out that after annealing the sticky ends of the Xba1 and Spe1 sites, the scar created had a stop codon TAG (UAG) that was in frame with the suppressed codon. When the Xba1 site on insert anneals to the Spe1 site on the plasmid (the green portions), we get a scar that reads TACTAG.
[[Image:screen-capture.png]]

For the afternoon, Olivia Ho-Shing and I finished assembly of the oligos of the suppressor tRNA codes. We assembled the document below:

http://gcat.davidson.edu/GcatWiki/images/5/5e/TRNAoligostoOrder.doc

We were assigned tasks of creating "cartoons" to illustrate the phenomenon that had occurred. No one could successfully do this however, because we could not understand how the piece had formed. The parts registry website provided wrong information on the restriction site of Spe1 which slowed our efficiency. This project turned into an effort to try to find two other different restriction enzymes that have complementary sticky ends other than Xba1 and Spe1. We want to get rid oc the scar that contains TACTAG.

We ended up deciding that a hybrid of two ideas was needed: restriction enzymes would need to be used to make several reporter gene sequences with different beginnings dependending on the suppressor tRNA sequence needed.

==Friday, June 5, 2009==

Our goal for this morning was to discuss the hybrid PCR approach to assemble the part that comes before the gene we wanted to express. We decided that we would have a BioBrick prefix and a 5mer followed by a sequence of nucleotides whose identity was up to us. Using PCR we would get the second double strand if we have the following:

BioBrick Prefix--ATG--5mer--gene until we get to restriction site

The PCR primer would attach to the ATG and the 5mer and would replicate the complementary base pairs to make a double stranded DNA sequence. As several of us were confused about PCR methods, we looked at the online link provided by Alyndria Thompson's link to the PCR interactive website which was very helpful. We also did some research to discover that the restriction enzyme BseRI (which is contained in the YFP that we wanted to alter) is not compatible with any other restriction enzyme. This is good because we did not want to plasmid containing this gene to close up with Xba1 once we digested it.

The team experienced some confusion with the hybrid idea and spoke to Clif Davis of Missouri Western University on the phone. He told us that there will be one part that is a fragment of the beginning of some nucleotides as a restriction cleaving site, ATG, the logical clause, and the start of the YFP gene that will be placed into a plasmid containing a space. To the left of the space will be the BioBrick Prefix and the right of the space will contain the rest of the genes in the YFP. Therefore, we decided not to alter any BioBrick ends and just look at restriction sites at the beginning of reporter proteins. The small fragment that will be placed into the plasmid will be made by PCR. The PCR will attach a primer onto the ATG and 5mer and replicate.

While we were discussing this, we expanded upon the idea that we can do whatever we want with the "suffix" portion between the 5mer and the beginning of YFP. We decided it would be best to remove ATG and add the first 5 amino acids and the restriction enzyme site. Along with this, if we have the 5mer coding for a certain amino acid (ie methionine) we could replace another codon that codes for methionine WITH that 5mer so long as this codon is contained within the first 15 nt. This way, we would not be altering the protein.

Olivia Ho-Shing came up with an idea of cascading PCR effect for longer logical clauses. Post translational modification of the protein was another one of her ideas. This applies because although we may code for the protein in a given way, the protein may modify or fix itself up to perform a slightly different function in its tertiary form.

Dr. Campbell suggested that we can look up restriction enzymes 400-500 base pairs into the reporter protein we would like to use; however, we have more flexibility in our procedure if we choose more "hardy" cutters. So instead, we could cut with EcoRI instead of with Xba1.

During the afternoon, we set about three tasks:
1) Which primers to use
2) Which promoters to use
3) Which restriction enzymes and how they will determine use of reporter proteins

Olivia Ho-Shing and I began looking up which restriction enzymes were strong so we could base our reporters on that. We began with Tet and RFP since those were the two reporters we had started out with choosing. I found a website that provided enzyme cleavage efficiency based upon the proximity of the site to the end of the PCR fragment that contains it. We discovered the following based upon the attached documents:

http://gcat.davidson.edu/GcatWiki/images/8/8f/Restriction_Enzyme_Cleavage_Efficiency.doc
http://gcat.davidson.edu/GcatWiki/images/9/9a/07Jun09_RE_Cutting_Efficiency_oh.doc

We decided that these two reporters contained efficient enzymes in a good quantity to choose from. On Monday, we will look through these enzymes once more and finalize which enzymes we would like to use with RFP and Tet.

==Sunday, June 7, 2009==

We did preparations for the mini prep this evening. The following notes are courtesy of Olivia Ho-Shing.

1. Sterically transfer ~2mL LB+AMP (AMP are ampicillin resistant plasmids) *Never put fluid back into bottle once it has entered pipette*
2. Use forceps to grab toothpick, scrap some frozen cells, rub in tube with top

'''Tubes'''

S03511 (2)

K091111

B0030

I715039

K091112

I715039

J31007 (2)

B0034

E1010

S03710

==Monday, June 8, 2009==

We reviewed the PCR process this morning as well as the restriction enzyme cleavage process.

Then, Leland Taylor, Olivia Ho-Shing and I all worked on converting the tRNA oligos we made to the template that Missouri Western used. The oligos we had previously made were different from the ones that Dr. Anderson had originally emailed us because further communication between Dr. Anderson and Missouri Western resulted in a better template.

We tried several times to minimize the number of oligos while keeping their individual lengths under 80bp. We managed to do this in the end by creating 7 oligos, 2 of which are variable between the suppressor tRNAs.
We reviewed the following document and finalized the oligos that we needed to order for the frameshift suppressor tRNAs. We decided to keep both the 10bp and 9bp anticodons of CCAUC.

I also looked up the amino acids that corresponded to the FS (frameshift suppressor) tRNAs for future use:

1. CUAGU = Leu
2. CCCUC = Pro
3. CGGUC = Arg
4. CCACU = Pro
5. CCAUC* = Pro
*9 and 10 bp anticodons

Also, we have decided to use NcoI as the restriction enzyme for RFP. This cleavage site occurs 419 bp into the gene. We have decided to use BamHI as the enzyme for Tetracycline Resistance and the cleavage site occurs 290bp into the gene. Both of these enzymes are very powerful and hardy.

We also looked over Missouri Western's tRNA oligos and primer oligos which we approved.
Our oligos were also approved by Missouri Western and Dr. Campbell ordered them while we supervised.

In the afternoon we did the miniprep. The mini prep directions were as appear in the link below.

http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_MiniPrep.html

We then worked with the Nano Prep to dilute our solution of DNA to an absorption of 1.5 and then we made labels for the tubes. The chart below shows relevant information we obtained from the NanoDrop for each of the reporter plasmids.

[[Image: Screen-capture-6.png]]

==Tuesday, June 9, 2009==

'''Preparations for restriction enzymes digestion of plasmids'''

Purpose: To determine the size of the plasmid inserts and ensure that plasmids are what we extracted from the cells during the mini-prep.

Things we need:
DNA ~3uL
Buffer 10x (Want to dilute to 1x) so add 2uL
Enzyme 1 ~1uL
Enzme 2 ~1uL
Water ~13uL
Final Volunter about 20 uL.

Need to choose the right buffer for the restriction enzymes. All buffers come in 10x (10 times more concentrated than what you want it to be when you use it). [http://gcat.davidson.edu/GcatWiki/index.php/Davidson_Missouri_W/Double_Digest_Guide Buffer Website]

Total volume of enzymes has to be 10% or less than final volume (most volume 2uL). Barely submerge the pipet tip into the solution when pipeting the enzymes.
Star (*) activity: Restriction enzyme stops cutting specifically and starts cutting everywhere.

DNA can't be seen by itself so we add EtBr (the bigger the insert, the more will bind) so bigger strands appear brighter. samples.

The order of entry doesn't matter, but make sure to enter the enzymes last.

Will have lots of labeled tubes and will put 3uL of appropriate plasmids into the tubes.
In the meantime, we'll make a "Master Mix" of all the other ingredients in a bigger tube. Always make enough volume for one extra tube.

Enzymes usually have an optimal temperature of 37C. We will let this reaction go for an hour and then we will run it on a gel.

'''Gel Electrophoresis Protocol'''

Website online for ideal gel concentration for resolving different sizes of molecules [http://gcat.davidson.edu/iGEM08/gelwebsite/gelwebsite.html Gel Concentration Website].
See protocol on the Davidson/Missouri Western Wookie for running molecules in the gel and how to digest with restriction enzymes.

'''What's Next?'''

1. 5' end additions
a. Using PCR
b. Cut at the restriction sites EcoR1 and either Bam H1 or Nco1
c. Cut plasmid and reporter with same restriction enzymes as above
d. Run a gel and purify "keeper" DNA (Ligate transform, screen, sequence)
e. Then, we take the "keeper" DNA and combine it with the insert from part

We can have steps d and e ready in advance.

2. tRNAs
a. Assemble the oligos into genes (Requires lots of calculations beforehand, but the actual steps )
b. Plasmids cut up with EcoR1 and Pst1 '''WE CAN USE THE "WASTE" PLASMID FROM THE RESTRICTION ENZYME DIGEST'''
c. Gel purify the plasmids (Ligate transform, screen, sequence)

The following tables include Master Mix measurements, agarose gel concentrations and buffers.
http://gcat.davidson.edu/GcatWiki/images/0/08/Shamitapic3.png
http://gcat.davidson.edu/GcatWiki/images/d/d1/Shamitapic4.png
http://gcat.davidson.edu/GcatWiki/images/6/6f/Shamitapic5.png

We made the Master Mix with our DNA and put it in the incubator at 10:40 AM.

'''Preparing the Gels'''

Now, we will be making 2 gels, 1 at 0.8% and 2.5% (weight to volume percent). The gel volume is 60mL.

'''Definition of 1% solution:
1 gram of agarose/ 100mL buffer'''

So for a 0.8% solution: 0.8 grams agarose/100mL buffer AND we need 60mL (fixed volume for casts run) so multiply that by 0.6.
Final concentration:

.48 grams agarose/60mL buffer

1 uL of EtBr stock for 60mL of gel.
EtBr is in the buffer so it is constantly replenishing itself even though it is moving toward one end in order to make bands on the other end visible.

After Dr. Campbell's demonstration, we each helped prepare a 0.8% buffer solution using the agarose powder, buffer and EtBr. We mixed together the first two substances in a 200mL Erlenmeyer flask and heated it for 120 seconds in the microwave. We then added EtBr and poured the gel solution into the gel cast.

'''Agar Plate'''

After lunch, we grew some more cells for each of the 11 parts in an agar plate labeled "LBAMP-AMC-9 June" so that so would have extra cells that could be stored for up to 2 weeks.
We made small circles on the agar plate and numbered them 1-11. The numbers corresponded to the following parts. We put the plate in the incubator overnight to let the cells grow.

1. B0030 AMC
2. K091111
3. S03710
4. I715039 OEH
5. S03511 ORH
6. I715039 AST
7. B0030 SDP
8. K091112
9. J31007 LJH
10. J31007 REC
11. S03511

'''Unloading the Gels'''

The following shows which parts we entered in which gels:

[[Image: screen-capture-2.png]]

We unloaded the 2.5% agarose first and after observing the bands under a UV light, put the gel into the BioRad machine to observe the bands. The image below is of a Molecular Weight Marker that shows the number of bp in control fragments of DNA according to the distance they travel down the gel.

[[Image: screen-capture-1.png]]

We obtained the following printout for the first gel that was 2.5% Agarose.
[[Image: 2009-06-09_14hr_44min.jpg]]

The bands in the first well at the bottom are those of the molecular weight marker. We skipped the lane above (to the right) of the MWM and entered the rest of the inserts as described in the above table. A light band is visible for each of the 4 other wells. In Lanes 3 and 4 we concluded we are supposed to have approximately 45 bp because the insert is 15bp + 30 bp on either side of the insert (Xba1 and Spe1 restrictions sites + sticky ends + extra bases). We used the same logic for Lanes 5 and 6 which are supposed to contain approximately 86 base pairs.

One discrepancy in this gel, however, is that Lanes 5 and 6, which according to their labels respectively contain the LacIQ promoter and the Lac IQ1 promoter. According to the information online at the parts registry website, pLacIQ1 should be the same length as pLacIQ. The gels do not show this, but instead show that the sample in Lane 5 is smaller than that in Lane 6 because Lane 5 traveled further. Leland Taylor and Romina Clemente sent out an email to Pallavi Penumetcha to ask about this because she had written a paper about this.

We then unloaded the 0.8% agarose gel and then placed this in the BioRad machine under the same protocol as above. The following image is the 0.8% Agarose.

[[Image:2009-06-09_15hr_21min.jpg]]

We observed that the bands in Lane 1 (which is now at the top) are examples of high intensity bands. The plasmids contained in the original well are twice the intensity of the insert.

In Lane 5, however, we observed several different bands. This could be due to the extra DNA that was sitting on the top of the well. We decided to re-expose the gel to get better observations on Lanes 3 and 4 because the bands were not as intense. We anticipated seeing a band at about 250 bp because this was the size of the insert (plus extra bp). After re-exposing the gel, we did not find any difference and still did not see any bands in these lanes. We have decided to redo the gel on the part S03511 and so we prepared the master mix with that DNA and incubated it so that we could run the gel tomorrow.

We also cut out the "empty" plasmids from the gel so that we could use this as vectors for the coding sequence of the suppressor tRNAs.

Tomorrow we will be planning out specific tasks for people once the oligos arrive.

==Wednesday, June 10, 2009==

We began this morning by making the agarose gel with the same protocol as yesterday. This time, since we are just re-doing both S03511 inserts, we will have a 1.8% agarose gel.

'''OUTLINE: WHERE DO WE STAND?'''

Yesterday
a. Isolated empty plasmids
b. Verified most inserts
c. Waiting for:
>tRNA oligos
>Primers
a. Will amplify the reporter gene in the plasmid itself

Next?
a. When Primers Arrive
> Amplify the reporter genes with primers (PCR takes ~3 hours)
> Calculate how much reporter plasmid we will need (Start with 50x more than we actually will need)
> Cut amplified sequence (EcoR1 and BamH1/Nco1) then purify what we need
> Cut reporter plasmid (EcoR1 and BamH1/Nco1) then purify what we need
> Ligate amplified sequence and reporter plasmid
b. When tRNA oligos arrive
> Calculate how much of each oligo we will need (takes a while...)
> "Boil and cool" tRNA oligos together
> Ligate fragments (dsDNA) into plasmids we isolated yesterday (~5 minutes)
> Transform the plasmids (~20 minutes)

* LOOK THROUGH LIGATION PROTOCOL (How to assemble genes)
* LOOK THROUGH TRANSFORMATION PROTOCOL

''What we need to get done today'':
1. Prepare the control for the Tet Resistance by BioBricking the part pLac+RBS (S03511) with the TetA (J31007).
2. Check the part number for RFP and also for pLac+RBS+RFP
3. Read through ligation protocol
4. See how many nanograms of plasmid we will need for the tRNAs and for the inserts and multiply this number by 50.
> Check if we have enough plasmids from our mini-prep
5. Purify the plasmids from the gel

After we made this outline, Leland, Olivia and I analyzed the gel we ran this morning and did not see a more than one band in the 2 lanes that contained DNA from S03511 (image below). Since we did not see multiple bands in yesterday's gel either, we conclude that there is something wrong with the DNA rather than with the restriction enzymes that we used to cut it. We concluded that there is probably not an insert in this plasmid both the part and the plasmid are useless.

[[Image: 2009-06-10 10hr 30min.jpg]]

Now, we do not have an immediately accessible promoter. We have decided to order the pBAD promoter from Missouri Western and we will attach the RBS+Tet to this plasmid. Our insert for this will be the larger gene because it is easier to manipulate in the gel than the small gene.

Alyndria Thompson and Romina Clemente are figuring out the size of the plasmids including the reporter genes.

After lunch we performed several steps to isolate and purify the gel to get the empty plasmids will be the vectors for the tRNA DNA. Then, we used the NanoDrop to assess the purity of our DNA. We found that the plasmids were more pure than we had expected. The following values were obtained:

Abs 1.850
A-260 10 mm path 0.172
A-280 10 mm path 0.075
260/280 2.28
260/230 0.09

We then followed the protocol for preparing the agar plates. We put the plate with the agar solution in the Autoclave.

While the solution was in the autoclave, we discussed that we don't really need a promoter in front of RBS+Tet for the gene to be expressed because Tet expression is so strong that the gene would translate without a promoter. However, it would be a good idea to have the promoter because if it was possible to reinforce the translation of the reporter protein, we wanted to do that.

We found that there were some tubes in the lab fridge that contained the pBAD and pLac promoters with RBS. We decided to use these as the promoter and RBS for our experiments. The pLac promoter will be used for RFP and the pBad promoter used with Tet Resistance. We decided to use the pBad with Tet because it is weaker in comparison to pLac. As expression of the Tet gene can be toxic to a cell, we do not want to overexpress the gene with a stronger promoter.

Once the agar solution was sterilized in the Autoclave, we removed it, cooled it, and pipetted the antibiotic resistance into it. We then poured the solution into the plates, removed any bubbles, and let the gel form.

Alyndria Thompson and Romina Clemente concluded that we had more than enough recieving vectors for both the RFP and Tetracycline genes. Since we need 50 nanograms, it is wise to have at least 50 times that much that we can work with, so we need at least 2,500 ng.

RFP 1: 77.6 x 4 = 310.4 ng/uL
x 20 uL = 3,272 ng
RFP 2: 59.5 x 4 = 238 ng/uL
x 20 uL = 4,760 ng
Tet: 40.9 x 4 = 163.6 ng/uL
x 20 uL = 3,272 ng

According to these calculations we have more than 50 times what we need for both instances of RFP and Tet.

Tomorrow, we can prepare the Master Mix for the digestions of both reporter plamids and then add the restriction enzymes once they arrive. Then, we can follow through with the rest of the plans we had made above in the section titles "Next?".

==Thursday, June 11, 2009==

We began this morning by calculating how many uL we would need for each of the mixes for digesting our reporter plasmids. We decided to double the final volume of each mix to 40uL instead of 20uL. Since we knew the concentration of each plasmid and also knew we needed 2500 ng, we cross multiplied to find the uL of DNA we would need for each.

Concentration Calculations:
310.4 ng/1 uL = 2500ng/x uL
x = 8.05 uL

Here is a chart indicating how much of each we used for the 2 mixes.

[[Image: DoubledigestRFPTetR.png]]

On the Promega website, we found a list of compatible buffers for double digestions. Buffers E and H are compatible for the Tet plasmid and RFP plasmid digestions respectively. We fortunately have both buffers in stock.

[[Image:BamEcoR1.png]] [[Image:NcoEcoR1.png]]

We waited for our waited for the restriction enzymes to arrive and in the meantime, we transformed the plasmids with pLac+RBS, pBad, and pBad+RBS into ''E.coli'' cells. We want the ''E.coli'' cells to replicate the plasmids with these genes because we have a limited amount of genes. [http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_Transformation.html Zippy Plasmid Transformation]

Once our oligos and restriction enzymes arrived, we got to work right away!

'''PCR (Polymerase Chain Reaction)'''

We calculated how much plasmid we will need for the PCR. We decided to dilute the concentration of our plasmids a hundred fold so that we could get pipetable amounts.

''Change in Concentrations''
310.4 ng/uL x 1/100 (dilution factor) = 3.104 ng/uL (RFP)
163.6 ng/uL x 1/100 (dilution factor) = 0.164 ng/uL (Tet)

''Example shown with RFP''
3.104 ng/1 uL = 1ng/ x uL, x = .32
For Tet, x = .61

We also calculated how much of each primer we would need with the following equation:

C1V1 = C2V2
(100 uM) (x) = (1 uM)( 100 uL)
x = 1 uL

[[Image: screen-capture-8.png]]

Each of the 5 of us made had 2 tubes (one for each reporter protein) because there were 5 different forward primers.To each tube, we added the template DNA (either RFP or Tet), water, the respective reverse primer (either RFP or Tet), and the forward primer depending on which 5mer we had chosen. My 5mer was CCAUC. Finally, we added the Master Mix last because this contained the enzyme (DNA polymerase) that would amplify our desired sequence.

[[Image: Pcrtubes.png]]

We followed the temperature cycles so that PCR could occur. The annealing temperature needed to be 5C less than the smallest melting temperature of the primers. Since the reverse primer of RFP had a melting temp of 66C, we set the temperature of the 3rd cycle on 61C.

[[Image: Temperaturecycle.png]]

'''tRNA Oligo Assembly'''

We needed to calculate the concentration of each of the oligo before carrying out the assembly. The stock concentration of each of the oligo was 100 uM. Since we wanted to have 5uM of the oligo in the test tube, and we know that final volume of the test tube is 20 uL, we just cross multiplied to find the amount we needed to add to the tubes to get this concentration.

(100uM)x = (5uM)(20uL)
x = 1 uL

We added 1 uL of each oligo to the tubes.

We had six total tRNAs we needed to assemble so each of us took one FML (one person took two) and to each tube we added 1uL of each oligo for a total of 7uL, 2uL of buffer, and 11uL of water for a total volume of 20 uL. Five of the oligos were constant and 2 varied for each of the different tRNAs. So everyone pipetted the same 5 oligos and everyone also had 2 unique oligos to add. When all of the oligos were added and the tubes ready, we boiled the tubes at 50C for 5 minutes. We then allowed the tubes to remain in the water to cool very slowly overnight so that the annealing process could take place.

The last thing we did at the end of the day was finish off our plasmid digestion that we had begun in the morning. We added the buffer, DNA, water, and restriction enzymes and placed this in the incubator for overnight digestion.

'''To Do''':

1. Isolate our desired dsDNA from the PCR.
2. Verify our tRNA inserts and eventually transform these into plasmids.
3. Check our agar plates to see colonies that have replicated our pLac+RBS, pBAD+RBS, and pBAD genes.

==Friday, June 12, 2009==

This morning, we began by outlining the main items on today's agenda.

1. tRNAs: Ligation, Transformation
2. Digestion reporters (run all of it and gel purify)
3. Transformed cells
4. PCR: Gel run an aliquot to verify (5uL)

We used the equation given by the Ligation protocol to calculate how many nanograms of insert we would need. Initially, we started out calculated the MW average of the 9 and 10 anticodon loops of tRNAs which were 15828.23 ug/umols and 159016.6 ug/umols respectively. We then saw, however, that these would not be helpful values in the process. Instead we started out with the equation below to find the final ng of insert we would need for the ligation.

[[Insert: screen]]

X nanograms of insert = [(2)(144bp)(50 ng linearalized plasmid)]/2038bp
X = 7.0657 ng of insert

We calculated our initial concentration in ng/uL of insert as shown below.

[[Image: screen-capture-1.png]]

We wanted to find the amount of uL of insert we would need to use. We diluted the initial concentration 50x because we wanted a pipettable amount of insert. Since we knew we wanted the final amount of nanograms to be 7.0657, we calculated as shown below:

''DILUTION'': 555.48/50 = 11.012
''CALCULATION'': 11.012 ng/ 1uL = 7.065 ng/ x uL
x = .64 uL of insert

After we had done these calculations, half of the group started to carryout the ligation of the tRNA inserts into empty plasmids. We needed 7 ligations total: 6 were the tRNAs we needed, while the last one is a negative control. These are the final amounts of what we used:

[[Image: trnaligation.jpg]]

The other half of the group (which included me) prepared the agarose gels so that we could run gels for the digested reporters and the PCR inserts. We calculated that the digested reporters needed an agarose of 4% and the PCR reporters needed an agarose of 1.45%. We allowed this gel to form for about 45 minutes to an hour. We prepared both the gels and allowed them to run through lunch.
After lunch, we took pictures of our gels and noticed that the third lane in the PCR gel did not develop a band. The lane was supposed to contain the CUAGU insert for Tetracycline Resistance.

Insert GEL pictures here.

Romina Clemente and I decided to repeat this PCR using the TetResistance and RBS+TetResistance plasmids as templates. We let the PCR run overnight. We also verified that the digestion fragments (both the plasmid and the insert) on the other gel were the correct size. The plasmids, which are the brighter fragment in the gel with only 2 lanes, contain the plasmid base pairs along with the rest of our reporter genes. We isolated these plasmids from the gel today and stored them for purification on Monday.

In the meantime, other members of our lab had finished the ligation of the tRNA genes to the empty plasmids and were now transforming these into competent cells. They placed the agar plate with these cells in the fridge overnight.

We also observed the transformations that we had performed yesterday and noticed that I13453 did not form any colonies. J04500 and J3102 did form colonies.

To Do:
1. PCR: Dig, Ligate, Transform
Clean and concentration column, digest, clean and concentration column, ligate.
2. Gel purification of our digestion plasmids
3. Check if colonies have grown for our tRNAs

IGEM Notebook

2009-06-12T22:51:38Z

ShPunjabi: /* Friday, June 12, 2009 */

==Wednesday, June 3, 2009==

Romina Clemente and I are trying to find suitable reporter proteins to use. Yesterday, Leland Taylor and Alyndria Thompson were working on ways to insert the gene sequences into the plasmid. Upon seeing how they wanted to manipulated the reporter gene to include the logical clauses, we came up with a few criteria for the reporter genes we would use. The following criteria for genes are listed in order of the broadest aspect to look at to the narrowest aspect:
a) Doesn't contain restriction sites for the 4 restriction enzymes (EcoR1, Xbal, Spel, Pst1) used to cleave the Biobrick part out of the plasmid.
b) Contains 6 cutter restriction sites.
c) These restriction enzymes aren't blunt (cleave straight down at one spot).
d) These restriction sites are close to thge 5' (beginning) end of the sequence.
e) These enzymes are easiest to work with and cheapest.
We are finding the part numbers of the reporter genes we want to use (antibiotic resistance, fluorescence, LacZ) through our own GCAT because we know these ones work. We are then locating these parts on the parts registry [1] website. We copied and pasted the gene sequences we obtained from the registry onto the ApE software [2]. From here, we were able to generate a genetic map of each gene that outlined each restriction site that fit our criteria. We put each genetic map, alongside the part number used, into a Word document.

http://gcat.davidson.edu/GcatWiki/images/0/0e/Restriction_Site_Mapping_on_Reporter_Genes.doc

Later on in the day, we decided that the best way to test for suppression would involve placing the 5mers at the beginning of the reporter gene instead of inserting them into the reporter gene. To do this, we decided that the gene along with its start codon need to be expressed after the 5mer. We will insert a BioBrick plasmid with a 5mer and start codon that will be incorporated before the gene:
RBS-6-8nt-ATG-5mer--ATG-gene

Leland Taylor assembled the oligos that we will need for the BioBrick pieces.

In order to test whether the reporter protein would be expressed regardless of the 5mer being suppressed or not, we would need to remove the suppressor tRNAs from the cell.

Meanwhile, I helped figure out the coding sequence that we will need for the suppressor tRNAs that will bind to the 5mers. We decided that we would be using the following suppressor tRNAs on the Davidson side: CUAGU, CCCUC, CGGUC, CCAUC, and CCACU. We searched through several papers, with little result and finally emailed Dr. Christopher Anderson of UC Berkeley to request the DNA sequence. He emailed us back with a generic DNA code that we could use to create the tRNA; however, for each suppressor tRNA we would use, we would need to change base pairs in the anticodon loop.

The length of the tRNA is 92 nt. The anticodon loop is comprised of 9 nt. Once we have BioBricked the DNA sequence with 4 restriction sites and supplemental nucleotides, the length of the entire gene is 144 nucleotides. This is made up of the 92 nt +22 nt on each side of the sequence (44) + 4 nt on each side of the restriction ends (8).

At the end of the day, we decided to use tetracycline resistance and RFP as our reporter proteins for this tester experiment.

==Thursday, June 4, 2009==

This morning, I began the day by learning how to use the PEARL prgram. Olivia Ho-Shing sat with me and walked me through the program script. And then, Leland Taylor and Shashank Suresh had a problem they wanted us to solve. Leland explained that we need to make sure there is a stop codon in the gene if we fail to suppress the 5mer. The stop codon needs to be before we find another 5mer. They also wanted us to run through both the RFP and Tet Resistance genes to make sure that they did not contain any of the anticodon sequences from our 5 frameshift suppressor tRNAs so as to throw the translation out of frame. They further wanted us to confirm that if we do have successful suppression, there are no stop codons in the middle of the RFP and Tet Resistance genes. PEARL did not find any matches for the suppressor tRNAs within genes. We found 3 matches for stop codons UAA. Two of these occured at the end of the Tet and one at the end of RFP, where they should normally be to end translation into a protein.

Following this, Olivia Ho-Shing and I tried to determine how we can add BioBrick ends to our single stranded DNA sequences that code for the suppressor tRNAs. Our ultimate goal is to find complements for these single strands so they can be put into plasmids. We found the standard prefixes and suffixes for sequences that do not contain ATG (becuase this sequence is a functional RNA and will not be translated) and we placed these before and after the altered 92bp sequence that Dr. Anderson provided us with. The prefixes and suffixes are necessary because they are the extra nucleotides that will be mimicking a "restriction enzyme cutting site". The single strand appeared as below after the Bio Brick prefixes and suffixes were added:

22ntPREFIX--32nt--VARIABLE ANTICODON LOOP--51nt--21ntSUFFIX

We used the suppressor codon CUAGU as our example and placed its appropriate anticodon loop in the sequence. We put this into the lancelator that would give us the other strand as well as the oligos we would need for ideal construction of this double stranded DNA fragment to put into the plasmid. We recieved a total of 4 oligos, 2 of which were variable and 2 of which were constant for all 6 different suppressor tRNAs. Using this double strand, we simulated a restriction enzyme digestion by EcoRI and PstI. We took off the G at both ends of the top strand and then removed the first five nucleotides on each end of the bottom strand. This made the top strand longer than the bottom one. We then proceeded to take the two variable strands and changed the anticodon loop nt for each suppressor tRNA we used.

Around lunchtime, we encountered a problem with the BioBrick scar in the "ATG-5mer subpiece" of the test project. It turns out that after annealing the sticky ends of the Xba1 and Spe1 sites, the scar created had a stop codon TAG (UAG) that was in frame with the suppressed codon. When the Xba1 site on insert anneals to the Spe1 site on the plasmid (the green portions), we get a scar that reads TACTAG.
[[Image:screen-capture.png]]

For the afternoon, Olivia Ho-Shing and I finished assembly of the oligos of the suppressor tRNA codes. We assembled the document below:

http://gcat.davidson.edu/GcatWiki/images/5/5e/TRNAoligostoOrder.doc

We were assigned tasks of creating "cartoons" to illustrate the phenomenon that had occurred. No one could successfully do this however, because we could not understand how the piece had formed. The parts registry website provided wrong information on the restriction site of Spe1 which slowed our efficiency. This project turned into an effort to try to find two other different restriction enzymes that have complementary sticky ends other than Xba1 and Spe1. We want to get rid oc the scar that contains TACTAG.

We ended up deciding that a hybrid of two ideas was needed: restriction enzymes would need to be used to make several reporter gene sequences with different beginnings dependending on the suppressor tRNA sequence needed.

==Friday, June 5, 2009==

Our goal for this morning was to discuss the hybrid PCR approach to assemble the part that comes before the gene we wanted to express. We decided that we would have a BioBrick prefix and a 5mer followed by a sequence of nucleotides whose identity was up to us. Using PCR we would get the second double strand if we have the following:

BioBrick Prefix--ATG--5mer--gene until we get to restriction site

The PCR primer would attach to the ATG and the 5mer and would replicate the complementary base pairs to make a double stranded DNA sequence. As several of us were confused about PCR methods, we looked at the online link provided by Alyndria Thompson's link to the PCR interactive website which was very helpful. We also did some research to discover that the restriction enzyme BseRI (which is contained in the YFP that we wanted to alter) is not compatible with any other restriction enzyme. This is good because we did not want to plasmid containing this gene to close up with Xba1 once we digested it.

The team experienced some confusion with the hybrid idea and spoke to Clif Davis of Missouri Western University on the phone. He told us that there will be one part that is a fragment of the beginning of some nucleotides as a restriction cleaving site, ATG, the logical clause, and the start of the YFP gene that will be placed into a plasmid containing a space. To the left of the space will be the BioBrick Prefix and the right of the space will contain the rest of the genes in the YFP. Therefore, we decided not to alter any BioBrick ends and just look at restriction sites at the beginning of reporter proteins. The small fragment that will be placed into the plasmid will be made by PCR. The PCR will attach a primer onto the ATG and 5mer and replicate.

While we were discussing this, we expanded upon the idea that we can do whatever we want with the "suffix" portion between the 5mer and the beginning of YFP. We decided it would be best to remove ATG and add the first 5 amino acids and the restriction enzyme site. Along with this, if we have the 5mer coding for a certain amino acid (ie methionine) we could replace another codon that codes for methionine WITH that 5mer so long as this codon is contained within the first 15 nt. This way, we would not be altering the protein.

Olivia Ho-Shing came up with an idea of cascading PCR effect for longer logical clauses. Post translational modification of the protein was another one of her ideas. This applies because although we may code for the protein in a given way, the protein may modify or fix itself up to perform a slightly different function in its tertiary form.

Dr. Campbell suggested that we can look up restriction enzymes 400-500 base pairs into the reporter protein we would like to use; however, we have more flexibility in our procedure if we choose more "hardy" cutters. So instead, we could cut with EcoRI instead of with Xba1.

During the afternoon, we set about three tasks:
1) Which primers to use
2) Which promoters to use
3) Which restriction enzymes and how they will determine use of reporter proteins

Olivia Ho-Shing and I began looking up which restriction enzymes were strong so we could base our reporters on that. We began with Tet and RFP since those were the two reporters we had started out with choosing. I found a website that provided enzyme cleavage efficiency based upon the proximity of the site to the end of the PCR fragment that contains it. We discovered the following based upon the attached documents:

http://gcat.davidson.edu/GcatWiki/images/8/8f/Restriction_Enzyme_Cleavage_Efficiency.doc
http://gcat.davidson.edu/GcatWiki/images/9/9a/07Jun09_RE_Cutting_Efficiency_oh.doc

We decided that these two reporters contained efficient enzymes in a good quantity to choose from. On Monday, we will look through these enzymes once more and finalize which enzymes we would like to use with RFP and Tet.

==Sunday, June 7, 2009==

We did preparations for the mini prep this evening. The following notes are courtesy of Olivia Ho-Shing.

1. Sterically transfer ~2mL LB+AMP (AMP are ampicillin resistant plasmids) *Never put fluid back into bottle once it has entered pipette*
2. Use forceps to grab toothpick, scrap some frozen cells, rub in tube with top

'''Tubes'''

S03511 (2)

K091111

B0030

I715039

K091112

I715039

J31007 (2)

B0034

E1010

S03710

==Monday, June 8, 2009==

We reviewed the PCR process this morning as well as the restriction enzyme cleavage process.

Then, Leland Taylor, Olivia Ho-Shing and I all worked on converting the tRNA oligos we made to the template that Missouri Western used. The oligos we had previously made were different from the ones that Dr. Anderson had originally emailed us because further communication between Dr. Anderson and Missouri Western resulted in a better template.

We tried several times to minimize the number of oligos while keeping their individual lengths under 80bp. We managed to do this in the end by creating 7 oligos, 2 of which are variable between the suppressor tRNAs.
We reviewed the following document and finalized the oligos that we needed to order for the frameshift suppressor tRNAs. We decided to keep both the 10bp and 9bp anticodons of CCAUC.

I also looked up the amino acids that corresponded to the FS (frameshift suppressor) tRNAs for future use:

1. CUAGU = Leu
2. CCCUC = Pro
3. CGGUC = Arg
4. CCACU = Pro
5. CCAUC* = Pro
*9 and 10 bp anticodons

Also, we have decided to use NcoI as the restriction enzyme for RFP. This cleavage site occurs 419 bp into the gene. We have decided to use BamHI as the enzyme for Tetracycline Resistance and the cleavage site occurs 290bp into the gene. Both of these enzymes are very powerful and hardy.

We also looked over Missouri Western's tRNA oligos and primer oligos which we approved.
Our oligos were also approved by Missouri Western and Dr. Campbell ordered them while we supervised.

In the afternoon we did the miniprep. The mini prep directions were as appear in the link below.

http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_MiniPrep.html

We then worked with the Nano Prep to dilute our solution of DNA to an absorption of 1.5 and then we made labels for the tubes. The chart below shows relevant information we obtained from the NanoDrop for each of the reporter plasmids.

[[Image: Screen-capture-6.png]]

==Tuesday, June 9, 2009==

'''Preparations for restriction enzymes digestion of plasmids'''

Purpose: To determine the size of the plasmid inserts and ensure that plasmids are what we extracted from the cells during the mini-prep.

Things we need:
DNA ~3uL
Buffer 10x (Want to dilute to 1x) so add 2uL
Enzyme 1 ~1uL
Enzme 2 ~1uL
Water ~13uL
Final Volunter about 20 uL.

Need to choose the right buffer for the restriction enzymes. All buffers come in 10x (10 times more concentrated than what you want it to be when you use it). [http://gcat.davidson.edu/GcatWiki/index.php/Davidson_Missouri_W/Double_Digest_Guide Buffer Website]

Total volume of enzymes has to be 10% or less than final volume (most volume 2uL). Barely submerge the pipet tip into the solution when pipeting the enzymes.
Star (*) activity: Restriction enzyme stops cutting specifically and starts cutting everywhere.

DNA can't be seen by itself so we add EtBr (the bigger the insert, the more will bind) so bigger strands appear brighter. samples.

The order of entry doesn't matter, but make sure to enter the enzymes last.

Will have lots of labeled tubes and will put 3uL of appropriate plasmids into the tubes.
In the meantime, we'll make a "Master Mix" of all the other ingredients in a bigger tube. Always make enough volume for one extra tube.

Enzymes usually have an optimal temperature of 37C. We will let this reaction go for an hour and then we will run it on a gel.

'''Gel Electrophoresis Protocol'''

Website online for ideal gel concentration for resolving different sizes of molecules [http://gcat.davidson.edu/iGEM08/gelwebsite/gelwebsite.html Gel Concentration Website].
See protocol on the Davidson/Missouri Western Wookie for running molecules in the gel and how to digest with restriction enzymes.

'''What's Next?'''

1. 5' end additions
a. Using PCR
b. Cut at the restriction sites EcoR1 and either Bam H1 or Nco1
c. Cut plasmid and reporter with same restriction enzymes as above
d. Run a gel and purify "keeper" DNA (Ligate transform, screen, sequence)
e. Then, we take the "keeper" DNA and combine it with the insert from part

We can have steps d and e ready in advance.

2. tRNAs
a. Assemble the oligos into genes (Requires lots of calculations beforehand, but the actual steps )
b. Plasmids cut up with EcoR1 and Pst1 '''WE CAN USE THE "WASTE" PLASMID FROM THE RESTRICTION ENZYME DIGEST'''
c. Gel purify the plasmids (Ligate transform, screen, sequence)

The following tables include Master Mix measurements, agarose gel concentrations and buffers.
http://gcat.davidson.edu/GcatWiki/images/0/08/Shamitapic3.png
http://gcat.davidson.edu/GcatWiki/images/d/d1/Shamitapic4.png
http://gcat.davidson.edu/GcatWiki/images/6/6f/Shamitapic5.png

We made the Master Mix with our DNA and put it in the incubator at 10:40 AM.

'''Preparing the Gels'''

Now, we will be making 2 gels, 1 at 0.8% and 2.5% (weight to volume percent). The gel volume is 60mL.

'''Definition of 1% solution:
1 gram of agarose/ 100mL buffer'''

So for a 0.8% solution: 0.8 grams agarose/100mL buffer AND we need 60mL (fixed volume for casts run) so multiply that by 0.6.
Final concentration:

.48 grams agarose/60mL buffer

1 uL of EtBr stock for 60mL of gel.
EtBr is in the buffer so it is constantly replenishing itself even though it is moving toward one end in order to make bands on the other end visible.

After Dr. Campbell's demonstration, we each helped prepare a 0.8% buffer solution using the agarose powder, buffer and EtBr. We mixed together the first two substances in a 200mL Erlenmeyer flask and heated it for 120 seconds in the microwave. We then added EtBr and poured the gel solution into the gel cast.

'''Agar Plate'''

After lunch, we grew some more cells for each of the 11 parts in an agar plate labeled "LBAMP-AMC-9 June" so that so would have extra cells that could be stored for up to 2 weeks.
We made small circles on the agar plate and numbered them 1-11. The numbers corresponded to the following parts. We put the plate in the incubator overnight to let the cells grow.

1. B0030 AMC
2. K091111
3. S03710
4. I715039 OEH
5. S03511 ORH
6. I715039 AST
7. B0030 SDP
8. K091112
9. J31007 LJH
10. J31007 REC
11. S03511

'''Unloading the Gels'''

The following shows which parts we entered in which gels:

[[Image: screen-capture-2.png]]

We unloaded the 2.5% agarose first and after observing the bands under a UV light, put the gel into the BioRad machine to observe the bands. The image below is of a Molecular Weight Marker that shows the number of bp in control fragments of DNA according to the distance they travel down the gel.

[[Image: screen-capture-1.png]]

We obtained the following printout for the first gel that was 2.5% Agarose.
[[Image: 2009-06-09_14hr_44min.jpg]]

The bands in the first well at the bottom are those of the molecular weight marker. We skipped the lane above (to the right) of the MWM and entered the rest of the inserts as described in the above table. A light band is visible for each of the 4 other wells. In Lanes 3 and 4 we concluded we are supposed to have approximately 45 bp because the insert is 15bp + 30 bp on either side of the insert (Xba1 and Spe1 restrictions sites + sticky ends + extra bases). We used the same logic for Lanes 5 and 6 which are supposed to contain approximately 86 base pairs.

One discrepancy in this gel, however, is that Lanes 5 and 6, which according to their labels respectively contain the LacIQ promoter and the Lac IQ1 promoter. According to the information online at the parts registry website, pLacIQ1 should be the same length as pLacIQ. The gels do not show this, but instead show that the sample in Lane 5 is smaller than that in Lane 6 because Lane 5 traveled further. Leland Taylor and Romina Clemente sent out an email to Pallavi Penumetcha to ask about this because she had written a paper about this.

We then unloaded the 0.8% agarose gel and then placed this in the BioRad machine under the same protocol as above. The following image is the 0.8% Agarose.

[[Image:2009-06-09_15hr_21min.jpg]]

We observed that the bands in Lane 1 (which is now at the top) are examples of high intensity bands. The plasmids contained in the original well are twice the intensity of the insert.

In Lane 5, however, we observed several different bands. This could be due to the extra DNA that was sitting on the top of the well. We decided to re-expose the gel to get better observations on Lanes 3 and 4 because the bands were not as intense. We anticipated seeing a band at about 250 bp because this was the size of the insert (plus extra bp). After re-exposing the gel, we did not find any difference and still did not see any bands in these lanes. We have decided to redo the gel on the part S03511 and so we prepared the master mix with that DNA and incubated it so that we could run the gel tomorrow.

We also cut out the "empty" plasmids from the gel so that we could use this as vectors for the coding sequence of the suppressor tRNAs.

Tomorrow we will be planning out specific tasks for people once the oligos arrive.

==Wednesday, June 10, 2009==

We began this morning by making the agarose gel with the same protocol as yesterday. This time, since we are just re-doing both S03511 inserts, we will have a 1.8% agarose gel.

'''OUTLINE: WHERE DO WE STAND?'''

Yesterday
a. Isolated empty plasmids
b. Verified most inserts
c. Waiting for:
>tRNA oligos
>Primers
a. Will amplify the reporter gene in the plasmid itself

Next?
a. When Primers Arrive
> Amplify the reporter genes with primers (PCR takes ~3 hours)
> Calculate how much reporter plasmid we will need (Start with 50x more than we actually will need)
> Cut amplified sequence (EcoR1 and BamH1/Nco1) then purify what we need
> Cut reporter plasmid (EcoR1 and BamH1/Nco1) then purify what we need
> Ligate amplified sequence and reporter plasmid
b. When tRNA oligos arrive
> Calculate how much of each oligo we will need (takes a while...)
> "Boil and cool" tRNA oligos together
> Ligate fragments (dsDNA) into plasmids we isolated yesterday (~5 minutes)
> Transform the plasmids (~20 minutes)

* LOOK THROUGH LIGATION PROTOCOL (How to assemble genes)
* LOOK THROUGH TRANSFORMATION PROTOCOL

''What we need to get done today'':
1. Prepare the control for the Tet Resistance by BioBricking the part pLac+RBS (S03511) with the TetA (J31007).
2. Check the part number for RFP and also for pLac+RBS+RFP
3. Read through ligation protocol
4. See how many nanograms of plasmid we will need for the tRNAs and for the inserts and multiply this number by 50.
> Check if we have enough plasmids from our mini-prep
5. Purify the plasmids from the gel

After we made this outline, Leland, Olivia and I analyzed the gel we ran this morning and did not see a more than one band in the 2 lanes that contained DNA from S03511 (image below). Since we did not see multiple bands in yesterday's gel either, we conclude that there is something wrong with the DNA rather than with the restriction enzymes that we used to cut it. We concluded that there is probably not an insert in this plasmid both the part and the plasmid are useless.

[[Image: 2009-06-10 10hr 30min.jpg]]

Now, we do not have an immediately accessible promoter. We have decided to order the pBAD promoter from Missouri Western and we will attach the RBS+Tet to this plasmid. Our insert for this will be the larger gene because it is easier to manipulate in the gel than the small gene.

Alyndria Thompson and Romina Clemente are figuring out the size of the plasmids including the reporter genes.

After lunch we performed several steps to isolate and purify the gel to get the empty plasmids will be the vectors for the tRNA DNA. Then, we used the NanoDrop to assess the purity of our DNA. We found that the plasmids were more pure than we had expected. The following values were obtained:

Abs 1.850
A-260 10 mm path 0.172
A-280 10 mm path 0.075
260/280 2.28
260/230 0.09

We then followed the protocol for preparing the agar plates. We put the plate with the agar solution in the Autoclave.

While the solution was in the autoclave, we discussed that we don't really need a promoter in front of RBS+Tet for the gene to be expressed because Tet expression is so strong that the gene would translate without a promoter. However, it would be a good idea to have the promoter because if it was possible to reinforce the translation of the reporter protein, we wanted to do that.

We found that there were some tubes in the lab fridge that contained the pBAD and pLac promoters with RBS. We decided to use these as the promoter and RBS for our experiments. The pLac promoter will be used for RFP and the pBad promoter used with Tet Resistance. We decided to use the pBad with Tet because it is weaker in comparison to pLac. As expression of the Tet gene can be toxic to a cell, we do not want to overexpress the gene with a stronger promoter.

Once the agar solution was sterilized in the Autoclave, we removed it, cooled it, and pipetted the antibiotic resistance into it. We then poured the solution into the plates, removed any bubbles, and let the gel form.

Alyndria Thompson and Romina Clemente concluded that we had more than enough recieving vectors for both the RFP and Tetracycline genes. Since we need 50 nanograms, it is wise to have at least 50 times that much that we can work with, so we need at least 2,500 ng.

RFP 1: 77.6 x 4 = 310.4 ng/uL
x 20 uL = 3,272 ng
RFP 2: 59.5 x 4 = 238 ng/uL
x 20 uL = 4,760 ng
Tet: 40.9 x 4 = 163.6 ng/uL
x 20 uL = 3,272 ng

According to these calculations we have more than 50 times what we need for both instances of RFP and Tet.

Tomorrow, we can prepare the Master Mix for the digestions of both reporter plamids and then add the restriction enzymes once they arrive. Then, we can follow through with the rest of the plans we had made above in the section titles "Next?".

==Thursday, June 11, 2009==

We began this morning by calculating how many uL we would need for each of the mixes for digesting our reporter plasmids. We decided to double the final volume of each mix to 40uL instead of 20uL. Since we knew the concentration of each plasmid and also knew we needed 2500 ng, we cross multiplied to find the uL of DNA we would need for each.

Concentration Calculations:
310.4 ng/1 uL = 2500ng/x uL
x = 8.05 uL

Here is a chart indicating how much of each we used for the 2 mixes.

[[Image: DoubledigestRFPTetR.png]]

On the Promega website, we found a list of compatible buffers for double digestions. Buffers E and H are compatible for the Tet plasmid and RFP plasmid digestions respectively. We fortunately have both buffers in stock.

[[Image:BamEcoR1.png]] [[Image:NcoEcoR1.png]]

We waited for our waited for the restriction enzymes to arrive and in the meantime, we transformed the plasmids with pLac+RBS, pBad, and pBad+RBS into ''E.coli'' cells. We want the ''E.coli'' cells to replicate the plasmids with these genes because we have a limited amount of genes. [http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_Transformation.html Zippy Plasmid Transformation]

Once our oligos and restriction enzymes arrived, we got to work right away!

'''PCR (Polymerase Chain Reaction)'''

We calculated how much plasmid we will need for the PCR. We decided to dilute the concentration of our plasmids a hundred fold so that we could get pipetable amounts.

''Change in Concentrations''
310.4 ng/uL x 1/100 (dilution factor) = 3.104 ng/uL (RFP)
163.6 ng/uL x 1/100 (dilution factor) = 0.164 ng/uL (Tet)

''Example shown with RFP''
3.104 ng/1 uL = 1ng/ x uL, x = .32
For Tet, x = .61

We also calculated how much of each primer we would need with the following equation:

C1V1 = C2V2
(100 uM) (x) = (1 uM)( 100 uL)
x = 1 uL

[[Image: screen-capture-8.png]]

Each of the 5 of us made had 2 tubes (one for each reporter protein) because there were 5 different forward primers.To each tube, we added the template DNA (either RFP or Tet), water, the respective reverse primer (either RFP or Tet), and the forward primer depending on which 5mer we had chosen. My 5mer was CCAUC. Finally, we added the Master Mix last because this contained the enzyme (DNA polymerase) that would amplify our desired sequence.

[[Image: Pcrtubes.png]]

We followed the temperature cycles so that PCR could occur. The annealing temperature needed to be 5C less than the smallest melting temperature of the primers. Since the reverse primer of RFP had a melting temp of 66C, we set the temperature of the 3rd cycle on 61C.

[[Image: Temperaturecycle.png]]

'''tRNA Oligo Assembly'''

We needed to calculate the concentration of each of the oligo before carrying out the assembly. The stock concentration of each of the oligo was 100 uM. Since we wanted to have 5uM of the oligo in the test tube, and we know that final volume of the test tube is 20 uL, we just cross multiplied to find the amount we needed to add to the tubes to get this concentration.

(100uM)x = (5uM)(20uL)
x = 1 uL

We added 1 uL of each oligo to the tubes.

We had six total tRNAs we needed to assemble so each of us took one FML (one person took two) and to each tube we added 1uL of each oligo for a total of 7uL, 2uL of buffer, and 11uL of water for a total volume of 20 uL. Five of the oligos were constant and 2 varied for each of the different tRNAs. So everyone pipetted the same 5 oligos and everyone also had 2 unique oligos to add. When all of the oligos were added and the tubes ready, we boiled the tubes at 50C for 5 minutes. We then allowed the tubes to remain in the water to cool very slowly overnight so that the annealing process could take place.

The last thing we did at the end of the day was finish off our plasmid digestion that we had begun in the morning. We added the buffer, DNA, water, and restriction enzymes and placed this in the incubator for overnight digestion.

'''To Do''':

1. Isolate our desired dsDNA from the PCR.
2. Verify our tRNA inserts and eventually transform these into plasmids.
3. Check our agar plates to see colonies that have replicated our pLac+RBS, pBAD+RBS, and pBAD genes.

==Friday, June 12, 2009==

This morning, we began by outlining the main items on today's agenda.

1. tRNAs: Ligation, Transformation
2. Digestion reporters (run all of it and gel purify)
3. Transformed cells
4. PCR: Gel run an aliquot to verify (5uL)

We used the equation given by the Ligation protocol to calculate how many nanograms of insert we would need. Initially, we started out calculated the MW average of the 9 and 10 anticodon loops of tRNAs which were 15828.23 ug/umols and 159016.6 ug/umols respectively. We then saw, however, that these would not be helpful values in the process. Instead we started out with the equation below to find the final ng of insert we would need for the ligation.

[[Insert: screen-capture-2.png]]

X nanograms of insert = [(2)(144bp)(50 ng linearalized plasmid)]/2038bp
X = 7.0657 ng of insert

We calculated our initial concentration in ng/uL of insert as shown below.

[[Image: screen-capture-1.png]]

We wanted to find the amount of uL of insert we would need to use. We diluted the initial concentration 50x because we wanted a pipettable amount of insert. Since we knew we wanted the final amount of nanograms to be 7.0657, we calculated as shown below:

''DILUTION'': 555.48/50 = 11.012
''CALCULATION'': 11.012 ng/ 1uL = 7.065 ng/ x uL
x = .64 uL of insert

After we had done these calculations, half of the group started to carryout the ligation of the tRNA inserts into empty plasmids. We needed 7 ligations total: 6 were the tRNAs we needed, while the last one is a negative control. These are the final amounts of what we used:

[[Image: trnaligation.jpg]]

The other half of the group (which included me) prepared the agarose gels so that we could run gels for the digested reporters and the PCR inserts. We calculated that the digested reporters needed an agarose of 4% and the PCR reporters needed an agarose of 1.45%. We allowed this gel to form for about 45 minutes to an hour. We prepared both the gels and allowed them to run through lunch.
After lunch, we took pictures of our gels and noticed that the third lane in the PCR gel did not develop a band. The lane was supposed to contain the CUAGU insert for Tetracycline Resistance.

Insert GEL pictures here.

Romina Clemente and I decided to repeat this PCR using the TetResistance and RBS+TetResistance plasmids as templates. We let the PCR run overnight. We also verified that the digestion fragments (both the plasmid and the insert) on the other gel were the correct size. The plasmids, which are the brighter fragment in the gel with only 2 lanes, contain the plasmid base pairs along with the rest of our reporter genes. We isolated these plasmids from the gel today and stored them for purification on Monday.

In the meantime, other members of our lab had finished the ligation of the tRNA genes to the empty plasmids and were now transforming these into competent cells. They placed the agar plate with these cells in the fridge overnight.

We also observed the transformations that we had performed yesterday and noticed that I13453 did not form any colonies. J04500 and J3102 did form colonies.

To Do:
1. PCR: Dig, Ligate, Transform
Clean and concentration column, digest, clean and concentration column, ligate.
2. Gel purification of our digestion plasmids
3. Check if colonies have grown for our tRNAs

IGEM Notebook

2009-06-12T22:40:52Z

ShPunjabi: /* Friday, June 12, 2009 */

==Wednesday, June 3, 2009==

Romina Clemente and I are trying to find suitable reporter proteins to use. Yesterday, Leland Taylor and Alyndria Thompson were working on ways to insert the gene sequences into the plasmid. Upon seeing how they wanted to manipulated the reporter gene to include the logical clauses, we came up with a few criteria for the reporter genes we would use. The following criteria for genes are listed in order of the broadest aspect to look at to the narrowest aspect:
a) Doesn't contain restriction sites for the 4 restriction enzymes (EcoR1, Xbal, Spel, Pst1) used to cleave the Biobrick part out of the plasmid.
b) Contains 6 cutter restriction sites.
c) These restriction enzymes aren't blunt (cleave straight down at one spot).
d) These restriction sites are close to thge 5' (beginning) end of the sequence.
e) These enzymes are easiest to work with and cheapest.
We are finding the part numbers of the reporter genes we want to use (antibiotic resistance, fluorescence, LacZ) through our own GCAT because we know these ones work. We are then locating these parts on the parts registry [1] website. We copied and pasted the gene sequences we obtained from the registry onto the ApE software [2]. From here, we were able to generate a genetic map of each gene that outlined each restriction site that fit our criteria. We put each genetic map, alongside the part number used, into a Word document.

http://gcat.davidson.edu/GcatWiki/images/0/0e/Restriction_Site_Mapping_on_Reporter_Genes.doc

Later on in the day, we decided that the best way to test for suppression would involve placing the 5mers at the beginning of the reporter gene instead of inserting them into the reporter gene. To do this, we decided that the gene along with its start codon need to be expressed after the 5mer. We will insert a BioBrick plasmid with a 5mer and start codon that will be incorporated before the gene:
RBS-6-8nt-ATG-5mer--ATG-gene

Leland Taylor assembled the oligos that we will need for the BioBrick pieces.

In order to test whether the reporter protein would be expressed regardless of the 5mer being suppressed or not, we would need to remove the suppressor tRNAs from the cell.

Meanwhile, I helped figure out the coding sequence that we will need for the suppressor tRNAs that will bind to the 5mers. We decided that we would be using the following suppressor tRNAs on the Davidson side: CUAGU, CCCUC, CGGUC, CCAUC, and CCACU. We searched through several papers, with little result and finally emailed Dr. Christopher Anderson of UC Berkeley to request the DNA sequence. He emailed us back with a generic DNA code that we could use to create the tRNA; however, for each suppressor tRNA we would use, we would need to change base pairs in the anticodon loop.

The length of the tRNA is 92 nt. The anticodon loop is comprised of 9 nt. Once we have BioBricked the DNA sequence with 4 restriction sites and supplemental nucleotides, the length of the entire gene is 144 nucleotides. This is made up of the 92 nt +22 nt on each side of the sequence (44) + 4 nt on each side of the restriction ends (8).

At the end of the day, we decided to use tetracycline resistance and RFP as our reporter proteins for this tester experiment.

==Thursday, June 4, 2009==

This morning, I began the day by learning how to use the PEARL prgram. Olivia Ho-Shing sat with me and walked me through the program script. And then, Leland Taylor and Shashank Suresh had a problem they wanted us to solve. Leland explained that we need to make sure there is a stop codon in the gene if we fail to suppress the 5mer. The stop codon needs to be before we find another 5mer. They also wanted us to run through both the RFP and Tet Resistance genes to make sure that they did not contain any of the anticodon sequences from our 5 frameshift suppressor tRNAs so as to throw the translation out of frame. They further wanted us to confirm that if we do have successful suppression, there are no stop codons in the middle of the RFP and Tet Resistance genes. PEARL did not find any matches for the suppressor tRNAs within genes. We found 3 matches for stop codons UAA. Two of these occured at the end of the Tet and one at the end of RFP, where they should normally be to end translation into a protein.

Following this, Olivia Ho-Shing and I tried to determine how we can add BioBrick ends to our single stranded DNA sequences that code for the suppressor tRNAs. Our ultimate goal is to find complements for these single strands so they can be put into plasmids. We found the standard prefixes and suffixes for sequences that do not contain ATG (becuase this sequence is a functional RNA and will not be translated) and we placed these before and after the altered 92bp sequence that Dr. Anderson provided us with. The prefixes and suffixes are necessary because they are the extra nucleotides that will be mimicking a "restriction enzyme cutting site". The single strand appeared as below after the Bio Brick prefixes and suffixes were added:

22ntPREFIX--32nt--VARIABLE ANTICODON LOOP--51nt--21ntSUFFIX

We used the suppressor codon CUAGU as our example and placed its appropriate anticodon loop in the sequence. We put this into the lancelator that would give us the other strand as well as the oligos we would need for ideal construction of this double stranded DNA fragment to put into the plasmid. We recieved a total of 4 oligos, 2 of which were variable and 2 of which were constant for all 6 different suppressor tRNAs. Using this double strand, we simulated a restriction enzyme digestion by EcoRI and PstI. We took off the G at both ends of the top strand and then removed the first five nucleotides on each end of the bottom strand. This made the top strand longer than the bottom one. We then proceeded to take the two variable strands and changed the anticodon loop nt for each suppressor tRNA we used.

Around lunchtime, we encountered a problem with the BioBrick scar in the "ATG-5mer subpiece" of the test project. It turns out that after annealing the sticky ends of the Xba1 and Spe1 sites, the scar created had a stop codon TAG (UAG) that was in frame with the suppressed codon. When the Xba1 site on insert anneals to the Spe1 site on the plasmid (the green portions), we get a scar that reads TACTAG.
[[Image:screen-capture.png]]

For the afternoon, Olivia Ho-Shing and I finished assembly of the oligos of the suppressor tRNA codes. We assembled the document below:

http://gcat.davidson.edu/GcatWiki/images/5/5e/TRNAoligostoOrder.doc

We were assigned tasks of creating "cartoons" to illustrate the phenomenon that had occurred. No one could successfully do this however, because we could not understand how the piece had formed. The parts registry website provided wrong information on the restriction site of Spe1 which slowed our efficiency. This project turned into an effort to try to find two other different restriction enzymes that have complementary sticky ends other than Xba1 and Spe1. We want to get rid oc the scar that contains TACTAG.

We ended up deciding that a hybrid of two ideas was needed: restriction enzymes would need to be used to make several reporter gene sequences with different beginnings dependending on the suppressor tRNA sequence needed.

==Friday, June 5, 2009==

Our goal for this morning was to discuss the hybrid PCR approach to assemble the part that comes before the gene we wanted to express. We decided that we would have a BioBrick prefix and a 5mer followed by a sequence of nucleotides whose identity was up to us. Using PCR we would get the second double strand if we have the following:

BioBrick Prefix--ATG--5mer--gene until we get to restriction site

The PCR primer would attach to the ATG and the 5mer and would replicate the complementary base pairs to make a double stranded DNA sequence. As several of us were confused about PCR methods, we looked at the online link provided by Alyndria Thompson's link to the PCR interactive website which was very helpful. We also did some research to discover that the restriction enzyme BseRI (which is contained in the YFP that we wanted to alter) is not compatible with any other restriction enzyme. This is good because we did not want to plasmid containing this gene to close up with Xba1 once we digested it.

The team experienced some confusion with the hybrid idea and spoke to Clif Davis of Missouri Western University on the phone. He told us that there will be one part that is a fragment of the beginning of some nucleotides as a restriction cleaving site, ATG, the logical clause, and the start of the YFP gene that will be placed into a plasmid containing a space. To the left of the space will be the BioBrick Prefix and the right of the space will contain the rest of the genes in the YFP. Therefore, we decided not to alter any BioBrick ends and just look at restriction sites at the beginning of reporter proteins. The small fragment that will be placed into the plasmid will be made by PCR. The PCR will attach a primer onto the ATG and 5mer and replicate.

While we were discussing this, we expanded upon the idea that we can do whatever we want with the "suffix" portion between the 5mer and the beginning of YFP. We decided it would be best to remove ATG and add the first 5 amino acids and the restriction enzyme site. Along with this, if we have the 5mer coding for a certain amino acid (ie methionine) we could replace another codon that codes for methionine WITH that 5mer so long as this codon is contained within the first 15 nt. This way, we would not be altering the protein.

Olivia Ho-Shing came up with an idea of cascading PCR effect for longer logical clauses. Post translational modification of the protein was another one of her ideas. This applies because although we may code for the protein in a given way, the protein may modify or fix itself up to perform a slightly different function in its tertiary form.

Dr. Campbell suggested that we can look up restriction enzymes 400-500 base pairs into the reporter protein we would like to use; however, we have more flexibility in our procedure if we choose more "hardy" cutters. So instead, we could cut with EcoRI instead of with Xba1.

During the afternoon, we set about three tasks:
1) Which primers to use
2) Which promoters to use
3) Which restriction enzymes and how they will determine use of reporter proteins

Olivia Ho-Shing and I began looking up which restriction enzymes were strong so we could base our reporters on that. We began with Tet and RFP since those were the two reporters we had started out with choosing. I found a website that provided enzyme cleavage efficiency based upon the proximity of the site to the end of the PCR fragment that contains it. We discovered the following based upon the attached documents:

http://gcat.davidson.edu/GcatWiki/images/8/8f/Restriction_Enzyme_Cleavage_Efficiency.doc
http://gcat.davidson.edu/GcatWiki/images/9/9a/07Jun09_RE_Cutting_Efficiency_oh.doc

We decided that these two reporters contained efficient enzymes in a good quantity to choose from. On Monday, we will look through these enzymes once more and finalize which enzymes we would like to use with RFP and Tet.

==Sunday, June 7, 2009==

We did preparations for the mini prep this evening. The following notes are courtesy of Olivia Ho-Shing.

1. Sterically transfer ~2mL LB+AMP (AMP are ampicillin resistant plasmids) *Never put fluid back into bottle once it has entered pipette*
2. Use forceps to grab toothpick, scrap some frozen cells, rub in tube with top

'''Tubes'''

S03511 (2)

K091111

B0030

I715039

K091112

I715039

J31007 (2)

B0034

E1010

S03710

==Monday, June 8, 2009==

We reviewed the PCR process this morning as well as the restriction enzyme cleavage process.

Then, Leland Taylor, Olivia Ho-Shing and I all worked on converting the tRNA oligos we made to the template that Missouri Western used. The oligos we had previously made were different from the ones that Dr. Anderson had originally emailed us because further communication between Dr. Anderson and Missouri Western resulted in a better template.

We tried several times to minimize the number of oligos while keeping their individual lengths under 80bp. We managed to do this in the end by creating 7 oligos, 2 of which are variable between the suppressor tRNAs.
We reviewed the following document and finalized the oligos that we needed to order for the frameshift suppressor tRNAs. We decided to keep both the 10bp and 9bp anticodons of CCAUC.

I also looked up the amino acids that corresponded to the FS (frameshift suppressor) tRNAs for future use:

1. CUAGU = Leu
2. CCCUC = Pro
3. CGGUC = Arg
4. CCACU = Pro
5. CCAUC* = Pro
*9 and 10 bp anticodons

Also, we have decided to use NcoI as the restriction enzyme for RFP. This cleavage site occurs 419 bp into the gene. We have decided to use BamHI as the enzyme for Tetracycline Resistance and the cleavage site occurs 290bp into the gene. Both of these enzymes are very powerful and hardy.

We also looked over Missouri Western's tRNA oligos and primer oligos which we approved.
Our oligos were also approved by Missouri Western and Dr. Campbell ordered them while we supervised.

In the afternoon we did the miniprep. The mini prep directions were as appear in the link below.

http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_MiniPrep.html

We then worked with the Nano Prep to dilute our solution of DNA to an absorption of 1.5 and then we made labels for the tubes. The chart below shows relevant information we obtained from the NanoDrop for each of the reporter plasmids.

[[Image: Screen-capture-6.png]]

==Tuesday, June 9, 2009==

'''Preparations for restriction enzymes digestion of plasmids'''

Purpose: To determine the size of the plasmid inserts and ensure that plasmids are what we extracted from the cells during the mini-prep.

Things we need:
DNA ~3uL
Buffer 10x (Want to dilute to 1x) so add 2uL
Enzyme 1 ~1uL
Enzme 2 ~1uL
Water ~13uL
Final Volunter about 20 uL.

Need to choose the right buffer for the restriction enzymes. All buffers come in 10x (10 times more concentrated than what you want it to be when you use it). [http://gcat.davidson.edu/GcatWiki/index.php/Davidson_Missouri_W/Double_Digest_Guide Buffer Website]

Total volume of enzymes has to be 10% or less than final volume (most volume 2uL). Barely submerge the pipet tip into the solution when pipeting the enzymes.
Star (*) activity: Restriction enzyme stops cutting specifically and starts cutting everywhere.

DNA can't be seen by itself so we add EtBr (the bigger the insert, the more will bind) so bigger strands appear brighter. samples.

The order of entry doesn't matter, but make sure to enter the enzymes last.

Will have lots of labeled tubes and will put 3uL of appropriate plasmids into the tubes.
In the meantime, we'll make a "Master Mix" of all the other ingredients in a bigger tube. Always make enough volume for one extra tube.

Enzymes usually have an optimal temperature of 37C. We will let this reaction go for an hour and then we will run it on a gel.

'''Gel Electrophoresis Protocol'''

Website online for ideal gel concentration for resolving different sizes of molecules [http://gcat.davidson.edu/iGEM08/gelwebsite/gelwebsite.html Gel Concentration Website].
See protocol on the Davidson/Missouri Western Wookie for running molecules in the gel and how to digest with restriction enzymes.

'''What's Next?'''

1. 5' end additions
a. Using PCR
b. Cut at the restriction sites EcoR1 and either Bam H1 or Nco1
c. Cut plasmid and reporter with same restriction enzymes as above
d. Run a gel and purify "keeper" DNA (Ligate transform, screen, sequence)
e. Then, we take the "keeper" DNA and combine it with the insert from part

We can have steps d and e ready in advance.

2. tRNAs
a. Assemble the oligos into genes (Requires lots of calculations beforehand, but the actual steps )
b. Plasmids cut up with EcoR1 and Pst1 '''WE CAN USE THE "WASTE" PLASMID FROM THE RESTRICTION ENZYME DIGEST'''
c. Gel purify the plasmids (Ligate transform, screen, sequence)

The following tables include Master Mix measurements, agarose gel concentrations and buffers.
http://gcat.davidson.edu/GcatWiki/images/0/08/Shamitapic3.png
http://gcat.davidson.edu/GcatWiki/images/d/d1/Shamitapic4.png
http://gcat.davidson.edu/GcatWiki/images/6/6f/Shamitapic5.png

We made the Master Mix with our DNA and put it in the incubator at 10:40 AM.

'''Preparing the Gels'''

Now, we will be making 2 gels, 1 at 0.8% and 2.5% (weight to volume percent). The gel volume is 60mL.

'''Definition of 1% solution:
1 gram of agarose/ 100mL buffer'''

So for a 0.8% solution: 0.8 grams agarose/100mL buffer AND we need 60mL (fixed volume for casts run) so multiply that by 0.6.
Final concentration:

.48 grams agarose/60mL buffer

1 uL of EtBr stock for 60mL of gel.
EtBr is in the buffer so it is constantly replenishing itself even though it is moving toward one end in order to make bands on the other end visible.

After Dr. Campbell's demonstration, we each helped prepare a 0.8% buffer solution using the agarose powder, buffer and EtBr. We mixed together the first two substances in a 200mL Erlenmeyer flask and heated it for 120 seconds in the microwave. We then added EtBr and poured the gel solution into the gel cast.

'''Agar Plate'''

After lunch, we grew some more cells for each of the 11 parts in an agar plate labeled "LBAMP-AMC-9 June" so that so would have extra cells that could be stored for up to 2 weeks.
We made small circles on the agar plate and numbered them 1-11. The numbers corresponded to the following parts. We put the plate in the incubator overnight to let the cells grow.

1. B0030 AMC
2. K091111
3. S03710
4. I715039 OEH
5. S03511 ORH
6. I715039 AST
7. B0030 SDP
8. K091112
9. J31007 LJH
10. J31007 REC
11. S03511

'''Unloading the Gels'''

The following shows which parts we entered in which gels:

[[Image: screen-capture-2.png]]

We unloaded the 2.5% agarose first and after observing the bands under a UV light, put the gel into the BioRad machine to observe the bands. The image below is of a Molecular Weight Marker that shows the number of bp in control fragments of DNA according to the distance they travel down the gel.

[[Image: screen-capture-1.png]]

We obtained the following printout for the first gel that was 2.5% Agarose.
[[Image: 2009-06-09_14hr_44min.jpg]]

The bands in the first well at the bottom are those of the molecular weight marker. We skipped the lane above (to the right) of the MWM and entered the rest of the inserts as described in the above table. A light band is visible for each of the 4 other wells. In Lanes 3 and 4 we concluded we are supposed to have approximately 45 bp because the insert is 15bp + 30 bp on either side of the insert (Xba1 and Spe1 restrictions sites + sticky ends + extra bases). We used the same logic for Lanes 5 and 6 which are supposed to contain approximately 86 base pairs.

One discrepancy in this gel, however, is that Lanes 5 and 6, which according to their labels respectively contain the LacIQ promoter and the Lac IQ1 promoter. According to the information online at the parts registry website, pLacIQ1 should be the same length as pLacIQ. The gels do not show this, but instead show that the sample in Lane 5 is smaller than that in Lane 6 because Lane 5 traveled further. Leland Taylor and Romina Clemente sent out an email to Pallavi Penumetcha to ask about this because she had written a paper about this.

We then unloaded the 0.8% agarose gel and then placed this in the BioRad machine under the same protocol as above. The following image is the 0.8% Agarose.

[[Image:2009-06-09_15hr_21min.jpg]]

We observed that the bands in Lane 1 (which is now at the top) are examples of high intensity bands. The plasmids contained in the original well are twice the intensity of the insert.

In Lane 5, however, we observed several different bands. This could be due to the extra DNA that was sitting on the top of the well. We decided to re-expose the gel to get better observations on Lanes 3 and 4 because the bands were not as intense. We anticipated seeing a band at about 250 bp because this was the size of the insert (plus extra bp). After re-exposing the gel, we did not find any difference and still did not see any bands in these lanes. We have decided to redo the gel on the part S03511 and so we prepared the master mix with that DNA and incubated it so that we could run the gel tomorrow.

We also cut out the "empty" plasmids from the gel so that we could use this as vectors for the coding sequence of the suppressor tRNAs.

Tomorrow we will be planning out specific tasks for people once the oligos arrive.

==Wednesday, June 10, 2009==

We began this morning by making the agarose gel with the same protocol as yesterday. This time, since we are just re-doing both S03511 inserts, we will have a 1.8% agarose gel.

'''OUTLINE: WHERE DO WE STAND?'''

Yesterday
a. Isolated empty plasmids
b. Verified most inserts
c. Waiting for:
>tRNA oligos
>Primers
a. Will amplify the reporter gene in the plasmid itself

Next?
a. When Primers Arrive
> Amplify the reporter genes with primers (PCR takes ~3 hours)
> Calculate how much reporter plasmid we will need (Start with 50x more than we actually will need)
> Cut amplified sequence (EcoR1 and BamH1/Nco1) then purify what we need
> Cut reporter plasmid (EcoR1 and BamH1/Nco1) then purify what we need
> Ligate amplified sequence and reporter plasmid
b. When tRNA oligos arrive
> Calculate how much of each oligo we will need (takes a while...)
> "Boil and cool" tRNA oligos together
> Ligate fragments (dsDNA) into plasmids we isolated yesterday (~5 minutes)
> Transform the plasmids (~20 minutes)

* LOOK THROUGH LIGATION PROTOCOL (How to assemble genes)
* LOOK THROUGH TRANSFORMATION PROTOCOL

''What we need to get done today'':
1. Prepare the control for the Tet Resistance by BioBricking the part pLac+RBS (S03511) with the TetA (J31007).
2. Check the part number for RFP and also for pLac+RBS+RFP
3. Read through ligation protocol
4. See how many nanograms of plasmid we will need for the tRNAs and for the inserts and multiply this number by 50.
> Check if we have enough plasmids from our mini-prep
5. Purify the plasmids from the gel

After we made this outline, Leland, Olivia and I analyzed the gel we ran this morning and did not see a more than one band in the 2 lanes that contained DNA from S03511 (image below). Since we did not see multiple bands in yesterday's gel either, we conclude that there is something wrong with the DNA rather than with the restriction enzymes that we used to cut it. We concluded that there is probably not an insert in this plasmid both the part and the plasmid are useless.

[[Image: 2009-06-10 10hr 30min.jpg]]

Now, we do not have an immediately accessible promoter. We have decided to order the pBAD promoter from Missouri Western and we will attach the RBS+Tet to this plasmid. Our insert for this will be the larger gene because it is easier to manipulate in the gel than the small gene.

Alyndria Thompson and Romina Clemente are figuring out the size of the plasmids including the reporter genes.

After lunch we performed several steps to isolate and purify the gel to get the empty plasmids will be the vectors for the tRNA DNA. Then, we used the NanoDrop to assess the purity of our DNA. We found that the plasmids were more pure than we had expected. The following values were obtained:

Abs 1.850
A-260 10 mm path 0.172
A-280 10 mm path 0.075
260/280 2.28
260/230 0.09

We then followed the protocol for preparing the agar plates. We put the plate with the agar solution in the Autoclave.

While the solution was in the autoclave, we discussed that we don't really need a promoter in front of RBS+Tet for the gene to be expressed because Tet expression is so strong that the gene would translate without a promoter. However, it would be a good idea to have the promoter because if it was possible to reinforce the translation of the reporter protein, we wanted to do that.

We found that there were some tubes in the lab fridge that contained the pBAD and pLac promoters with RBS. We decided to use these as the promoter and RBS for our experiments. The pLac promoter will be used for RFP and the pBad promoter used with Tet Resistance. We decided to use the pBad with Tet because it is weaker in comparison to pLac. As expression of the Tet gene can be toxic to a cell, we do not want to overexpress the gene with a stronger promoter.

Once the agar solution was sterilized in the Autoclave, we removed it, cooled it, and pipetted the antibiotic resistance into it. We then poured the solution into the plates, removed any bubbles, and let the gel form.

Alyndria Thompson and Romina Clemente concluded that we had more than enough recieving vectors for both the RFP and Tetracycline genes. Since we need 50 nanograms, it is wise to have at least 50 times that much that we can work with, so we need at least 2,500 ng.

RFP 1: 77.6 x 4 = 310.4 ng/uL
x 20 uL = 3,272 ng
RFP 2: 59.5 x 4 = 238 ng/uL
x 20 uL = 4,760 ng
Tet: 40.9 x 4 = 163.6 ng/uL
x 20 uL = 3,272 ng

According to these calculations we have more than 50 times what we need for both instances of RFP and Tet.

Tomorrow, we can prepare the Master Mix for the digestions of both reporter plamids and then add the restriction enzymes once they arrive. Then, we can follow through with the rest of the plans we had made above in the section titles "Next?".

==Thursday, June 11, 2009==

We began this morning by calculating how many uL we would need for each of the mixes for digesting our reporter plasmids. We decided to double the final volume of each mix to 40uL instead of 20uL. Since we knew the concentration of each plasmid and also knew we needed 2500 ng, we cross multiplied to find the uL of DNA we would need for each.

Concentration Calculations:
310.4 ng/1 uL = 2500ng/x uL
x = 8.05 uL

Here is a chart indicating how much of each we used for the 2 mixes.

[[Image: DoubledigestRFPTetR.png]]

On the Promega website, we found a list of compatible buffers for double digestions. Buffers E and H are compatible for the Tet plasmid and RFP plasmid digestions respectively. We fortunately have both buffers in stock.

[[Image:BamEcoR1.png]] [[Image:NcoEcoR1.png]]

We waited for our waited for the restriction enzymes to arrive and in the meantime, we transformed the plasmids with pLac+RBS, pBad, and pBad+RBS into ''E.coli'' cells. We want the ''E.coli'' cells to replicate the plasmids with these genes because we have a limited amount of genes. [http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_Transformation.html Zippy Plasmid Transformation]

Once our oligos and restriction enzymes arrived, we got to work right away!

'''PCR (Polymerase Chain Reaction)'''

We calculated how much plasmid we will need for the PCR. We decided to dilute the concentration of our plasmids a hundred fold so that we could get pipetable amounts.

''Change in Concentrations''
310.4 ng/uL x 1/100 (dilution factor) = 3.104 ng/uL (RFP)
163.6 ng/uL x 1/100 (dilution factor) = 0.164 ng/uL (Tet)

''Example shown with RFP''
3.104 ng/1 uL = 1ng/ x uL, x = .32
For Tet, x = .61

We also calculated how much of each primer we would need with the following equation:

C1V1 = C2V2
(100 uM) (x) = (1 uM)( 100 uL)
x = 1 uL

[[Image: screen-capture-8.png]]

Each of the 5 of us made had 2 tubes (one for each reporter protein) because there were 5 different forward primers.To each tube, we added the template DNA (either RFP or Tet), water, the respective reverse primer (either RFP or Tet), and the forward primer depending on which 5mer we had chosen. My 5mer was CCAUC. Finally, we added the Master Mix last because this contained the enzyme (DNA polymerase) that would amplify our desired sequence.

[[Image: Pcrtubes.png]]

We followed the temperature cycles so that PCR could occur. The annealing temperature needed to be 5C less than the smallest melting temperature of the primers. Since the reverse primer of RFP had a melting temp of 66C, we set the temperature of the 3rd cycle on 61C.

[[Image: Temperaturecycle.png]]

'''tRNA Oligo Assembly'''

We needed to calculate the concentration of each of the oligo before carrying out the assembly. The stock concentration of each of the oligo was 100 uM. Since we wanted to have 5uM of the oligo in the test tube, and we know that final volume of the test tube is 20 uL, we just cross multiplied to find the amount we needed to add to the tubes to get this concentration.

(100uM)x = (5uM)(20uL)
x = 1 uL

We added 1 uL of each oligo to the tubes.

We had six total tRNAs we needed to assemble so each of us took one FML (one person took two) and to each tube we added 1uL of each oligo for a total of 7uL, 2uL of buffer, and 11uL of water for a total volume of 20 uL. Five of the oligos were constant and 2 varied for each of the different tRNAs. So everyone pipetted the same 5 oligos and everyone also had 2 unique oligos to add. When all of the oligos were added and the tubes ready, we boiled the tubes at 50C for 5 minutes. We then allowed the tubes to remain in the water to cool very slowly overnight so that the annealing process could take place.

The last thing we did at the end of the day was finish off our plasmid digestion that we had begun in the morning. We added the buffer, DNA, water, and restriction enzymes and placed this in the incubator for overnight digestion.

'''To Do''':

1. Isolate our desired dsDNA from the PCR.
2. Verify our tRNA inserts and eventually transform these into plasmids.
3. Check our agar plates to see colonies that have replicated our pLac+RBS, pBAD+RBS, and pBAD genes.

==Friday, June 12, 2009==

This morning, we began by outlining the main items on today's agenda.

1. tRNAs: Ligation, Transformation
2. Digestion reporters (run all of it and gel purify)
3. Transformed cells
4. PCR: Gel run an aliquot to verify (5uL)

We used the equation given by the Ligation protocol to calculate how many nanograms of insert we would need. Initially, we started out calculated the MW average of the 9 and 10 anticodon loops of tRNAs which were 15828.23 ug/umols and 159016.6 ug/umols respectively. We then saw, however, that these would not be helpful values in the process. Instead we started out with the equation below to find the final ng of insert we would need for the ligation.

[[Insert: screen-capture-2.png]]

X nanograms of insert = [(2)(144bp)(50 ng linearalized plasmid)]/2038bp
X = 7.0657 ng of insert

We calculated our initial concentration in ng/uL of insert as shown below.

[[Image: screen-capture-1.png]]

We wanted to find the amount of uL of insert we would need to use. We diluted the initial concentration 50x because we wanted a pipettable amount of insert. Since we knew we wanted the final amount of nanograms to be 7.0657, we calculated as shown below:

''DILUTION'': 555.48/50 = 11.012
''CALCULATION'': 11.012 ng/ 1uL = 7.065 ng/ x uL
x = .64 uL of insert

After we had done these calculations, half of the group started to carryout the ligation of the tRNA inserts into empty plasmids. We needed 7 ligations total: 6 were the tRNAs we needed, while the last one is a negative control.

The other half of the group (which included me) prepared the agarose gels so that we could run gels for the digested reporters and the PCR inserts. We calculated that the digested reporters needed an agarose of 4% and the PCR reporters needed an agarose of 1.45%. We allowed this gel to form for about 45 minutes to an hour.

We then observed the transformations that we had performed yesterday and noticed that I13453 did not form any colonies. J04500 and J3102 did form colonies.

IGEM Notebook

2009-06-12T22:23:19Z

ShPunjabi: /* Friday, June 12, 2009 */

==Wednesday, June 3, 2009==

Romina Clemente and I are trying to find suitable reporter proteins to use. Yesterday, Leland Taylor and Alyndria Thompson were working on ways to insert the gene sequences into the plasmid. Upon seeing how they wanted to manipulated the reporter gene to include the logical clauses, we came up with a few criteria for the reporter genes we would use. The following criteria for genes are listed in order of the broadest aspect to look at to the narrowest aspect:
a) Doesn't contain restriction sites for the 4 restriction enzymes (EcoR1, Xbal, Spel, Pst1) used to cleave the Biobrick part out of the plasmid.
b) Contains 6 cutter restriction sites.
c) These restriction enzymes aren't blunt (cleave straight down at one spot).
d) These restriction sites are close to thge 5' (beginning) end of the sequence.
e) These enzymes are easiest to work with and cheapest.
We are finding the part numbers of the reporter genes we want to use (antibiotic resistance, fluorescence, LacZ) through our own GCAT because we know these ones work. We are then locating these parts on the parts registry [1] website. We copied and pasted the gene sequences we obtained from the registry onto the ApE software [2]. From here, we were able to generate a genetic map of each gene that outlined each restriction site that fit our criteria. We put each genetic map, alongside the part number used, into a Word document.

http://gcat.davidson.edu/GcatWiki/images/0/0e/Restriction_Site_Mapping_on_Reporter_Genes.doc

Later on in the day, we decided that the best way to test for suppression would involve placing the 5mers at the beginning of the reporter gene instead of inserting them into the reporter gene. To do this, we decided that the gene along with its start codon need to be expressed after the 5mer. We will insert a BioBrick plasmid with a 5mer and start codon that will be incorporated before the gene:
RBS-6-8nt-ATG-5mer--ATG-gene

Leland Taylor assembled the oligos that we will need for the BioBrick pieces.

In order to test whether the reporter protein would be expressed regardless of the 5mer being suppressed or not, we would need to remove the suppressor tRNAs from the cell.

Meanwhile, I helped figure out the coding sequence that we will need for the suppressor tRNAs that will bind to the 5mers. We decided that we would be using the following suppressor tRNAs on the Davidson side: CUAGU, CCCUC, CGGUC, CCAUC, and CCACU. We searched through several papers, with little result and finally emailed Dr. Christopher Anderson of UC Berkeley to request the DNA sequence. He emailed us back with a generic DNA code that we could use to create the tRNA; however, for each suppressor tRNA we would use, we would need to change base pairs in the anticodon loop.

The length of the tRNA is 92 nt. The anticodon loop is comprised of 9 nt. Once we have BioBricked the DNA sequence with 4 restriction sites and supplemental nucleotides, the length of the entire gene is 144 nucleotides. This is made up of the 92 nt +22 nt on each side of the sequence (44) + 4 nt on each side of the restriction ends (8).

At the end of the day, we decided to use tetracycline resistance and RFP as our reporter proteins for this tester experiment.

==Thursday, June 4, 2009==

This morning, I began the day by learning how to use the PEARL prgram. Olivia Ho-Shing sat with me and walked me through the program script. And then, Leland Taylor and Shashank Suresh had a problem they wanted us to solve. Leland explained that we need to make sure there is a stop codon in the gene if we fail to suppress the 5mer. The stop codon needs to be before we find another 5mer. They also wanted us to run through both the RFP and Tet Resistance genes to make sure that they did not contain any of the anticodon sequences from our 5 frameshift suppressor tRNAs so as to throw the translation out of frame. They further wanted us to confirm that if we do have successful suppression, there are no stop codons in the middle of the RFP and Tet Resistance genes. PEARL did not find any matches for the suppressor tRNAs within genes. We found 3 matches for stop codons UAA. Two of these occured at the end of the Tet and one at the end of RFP, where they should normally be to end translation into a protein.

Following this, Olivia Ho-Shing and I tried to determine how we can add BioBrick ends to our single stranded DNA sequences that code for the suppressor tRNAs. Our ultimate goal is to find complements for these single strands so they can be put into plasmids. We found the standard prefixes and suffixes for sequences that do not contain ATG (becuase this sequence is a functional RNA and will not be translated) and we placed these before and after the altered 92bp sequence that Dr. Anderson provided us with. The prefixes and suffixes are necessary because they are the extra nucleotides that will be mimicking a "restriction enzyme cutting site". The single strand appeared as below after the Bio Brick prefixes and suffixes were added:

22ntPREFIX--32nt--VARIABLE ANTICODON LOOP--51nt--21ntSUFFIX

We used the suppressor codon CUAGU as our example and placed its appropriate anticodon loop in the sequence. We put this into the lancelator that would give us the other strand as well as the oligos we would need for ideal construction of this double stranded DNA fragment to put into the plasmid. We recieved a total of 4 oligos, 2 of which were variable and 2 of which were constant for all 6 different suppressor tRNAs. Using this double strand, we simulated a restriction enzyme digestion by EcoRI and PstI. We took off the G at both ends of the top strand and then removed the first five nucleotides on each end of the bottom strand. This made the top strand longer than the bottom one. We then proceeded to take the two variable strands and changed the anticodon loop nt for each suppressor tRNA we used.

Around lunchtime, we encountered a problem with the BioBrick scar in the "ATG-5mer subpiece" of the test project. It turns out that after annealing the sticky ends of the Xba1 and Spe1 sites, the scar created had a stop codon TAG (UAG) that was in frame with the suppressed codon. When the Xba1 site on insert anneals to the Spe1 site on the plasmid (the green portions), we get a scar that reads TACTAG.
[[Image:screen-capture.png]]

For the afternoon, Olivia Ho-Shing and I finished assembly of the oligos of the suppressor tRNA codes. We assembled the document below:

http://gcat.davidson.edu/GcatWiki/images/5/5e/TRNAoligostoOrder.doc

We were assigned tasks of creating "cartoons" to illustrate the phenomenon that had occurred. No one could successfully do this however, because we could not understand how the piece had formed. The parts registry website provided wrong information on the restriction site of Spe1 which slowed our efficiency. This project turned into an effort to try to find two other different restriction enzymes that have complementary sticky ends other than Xba1 and Spe1. We want to get rid oc the scar that contains TACTAG.

We ended up deciding that a hybrid of two ideas was needed: restriction enzymes would need to be used to make several reporter gene sequences with different beginnings dependending on the suppressor tRNA sequence needed.

==Friday, June 5, 2009==

Our goal for this morning was to discuss the hybrid PCR approach to assemble the part that comes before the gene we wanted to express. We decided that we would have a BioBrick prefix and a 5mer followed by a sequence of nucleotides whose identity was up to us. Using PCR we would get the second double strand if we have the following:

BioBrick Prefix--ATG--5mer--gene until we get to restriction site

The PCR primer would attach to the ATG and the 5mer and would replicate the complementary base pairs to make a double stranded DNA sequence. As several of us were confused about PCR methods, we looked at the online link provided by Alyndria Thompson's link to the PCR interactive website which was very helpful. We also did some research to discover that the restriction enzyme BseRI (which is contained in the YFP that we wanted to alter) is not compatible with any other restriction enzyme. This is good because we did not want to plasmid containing this gene to close up with Xba1 once we digested it.

The team experienced some confusion with the hybrid idea and spoke to Clif Davis of Missouri Western University on the phone. He told us that there will be one part that is a fragment of the beginning of some nucleotides as a restriction cleaving site, ATG, the logical clause, and the start of the YFP gene that will be placed into a plasmid containing a space. To the left of the space will be the BioBrick Prefix and the right of the space will contain the rest of the genes in the YFP. Therefore, we decided not to alter any BioBrick ends and just look at restriction sites at the beginning of reporter proteins. The small fragment that will be placed into the plasmid will be made by PCR. The PCR will attach a primer onto the ATG and 5mer and replicate.

While we were discussing this, we expanded upon the idea that we can do whatever we want with the "suffix" portion between the 5mer and the beginning of YFP. We decided it would be best to remove ATG and add the first 5 amino acids and the restriction enzyme site. Along with this, if we have the 5mer coding for a certain amino acid (ie methionine) we could replace another codon that codes for methionine WITH that 5mer so long as this codon is contained within the first 15 nt. This way, we would not be altering the protein.

Olivia Ho-Shing came up with an idea of cascading PCR effect for longer logical clauses. Post translational modification of the protein was another one of her ideas. This applies because although we may code for the protein in a given way, the protein may modify or fix itself up to perform a slightly different function in its tertiary form.

Dr. Campbell suggested that we can look up restriction enzymes 400-500 base pairs into the reporter protein we would like to use; however, we have more flexibility in our procedure if we choose more "hardy" cutters. So instead, we could cut with EcoRI instead of with Xba1.

During the afternoon, we set about three tasks:
1) Which primers to use
2) Which promoters to use
3) Which restriction enzymes and how they will determine use of reporter proteins

Olivia Ho-Shing and I began looking up which restriction enzymes were strong so we could base our reporters on that. We began with Tet and RFP since those were the two reporters we had started out with choosing. I found a website that provided enzyme cleavage efficiency based upon the proximity of the site to the end of the PCR fragment that contains it. We discovered the following based upon the attached documents:

http://gcat.davidson.edu/GcatWiki/images/8/8f/Restriction_Enzyme_Cleavage_Efficiency.doc
http://gcat.davidson.edu/GcatWiki/images/9/9a/07Jun09_RE_Cutting_Efficiency_oh.doc

We decided that these two reporters contained efficient enzymes in a good quantity to choose from. On Monday, we will look through these enzymes once more and finalize which enzymes we would like to use with RFP and Tet.

==Sunday, June 7, 2009==

We did preparations for the mini prep this evening. The following notes are courtesy of Olivia Ho-Shing.

1. Sterically transfer ~2mL LB+AMP (AMP are ampicillin resistant plasmids) *Never put fluid back into bottle once it has entered pipette*
2. Use forceps to grab toothpick, scrap some frozen cells, rub in tube with top

'''Tubes'''

S03511 (2)

K091111

B0030

I715039

K091112

I715039

J31007 (2)

B0034

E1010

S03710

==Monday, June 8, 2009==

We reviewed the PCR process this morning as well as the restriction enzyme cleavage process.

Then, Leland Taylor, Olivia Ho-Shing and I all worked on converting the tRNA oligos we made to the template that Missouri Western used. The oligos we had previously made were different from the ones that Dr. Anderson had originally emailed us because further communication between Dr. Anderson and Missouri Western resulted in a better template.

We tried several times to minimize the number of oligos while keeping their individual lengths under 80bp. We managed to do this in the end by creating 7 oligos, 2 of which are variable between the suppressor tRNAs.
We reviewed the following document and finalized the oligos that we needed to order for the frameshift suppressor tRNAs. We decided to keep both the 10bp and 9bp anticodons of CCAUC.

I also looked up the amino acids that corresponded to the FS (frameshift suppressor) tRNAs for future use:

1. CUAGU = Leu
2. CCCUC = Pro
3. CGGUC = Arg
4. CCACU = Pro
5. CCAUC* = Pro
*9 and 10 bp anticodons

Also, we have decided to use NcoI as the restriction enzyme for RFP. This cleavage site occurs 419 bp into the gene. We have decided to use BamHI as the enzyme for Tetracycline Resistance and the cleavage site occurs 290bp into the gene. Both of these enzymes are very powerful and hardy.

We also looked over Missouri Western's tRNA oligos and primer oligos which we approved.
Our oligos were also approved by Missouri Western and Dr. Campbell ordered them while we supervised.

In the afternoon we did the miniprep. The mini prep directions were as appear in the link below.

http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_MiniPrep.html

We then worked with the Nano Prep to dilute our solution of DNA to an absorption of 1.5 and then we made labels for the tubes. The chart below shows relevant information we obtained from the NanoDrop for each of the reporter plasmids.

[[Image: Screen-capture-6.png]]

==Tuesday, June 9, 2009==

'''Preparations for restriction enzymes digestion of plasmids'''

Purpose: To determine the size of the plasmid inserts and ensure that plasmids are what we extracted from the cells during the mini-prep.

Things we need:
DNA ~3uL
Buffer 10x (Want to dilute to 1x) so add 2uL
Enzyme 1 ~1uL
Enzme 2 ~1uL
Water ~13uL
Final Volunter about 20 uL.

Need to choose the right buffer for the restriction enzymes. All buffers come in 10x (10 times more concentrated than what you want it to be when you use it). [http://gcat.davidson.edu/GcatWiki/index.php/Davidson_Missouri_W/Double_Digest_Guide Buffer Website]

Total volume of enzymes has to be 10% or less than final volume (most volume 2uL). Barely submerge the pipet tip into the solution when pipeting the enzymes.
Star (*) activity: Restriction enzyme stops cutting specifically and starts cutting everywhere.

DNA can't be seen by itself so we add EtBr (the bigger the insert, the more will bind) so bigger strands appear brighter. samples.

The order of entry doesn't matter, but make sure to enter the enzymes last.

Will have lots of labeled tubes and will put 3uL of appropriate plasmids into the tubes.
In the meantime, we'll make a "Master Mix" of all the other ingredients in a bigger tube. Always make enough volume for one extra tube.

Enzymes usually have an optimal temperature of 37C. We will let this reaction go for an hour and then we will run it on a gel.

'''Gel Electrophoresis Protocol'''

Website online for ideal gel concentration for resolving different sizes of molecules [http://gcat.davidson.edu/iGEM08/gelwebsite/gelwebsite.html Gel Concentration Website].
See protocol on the Davidson/Missouri Western Wookie for running molecules in the gel and how to digest with restriction enzymes.

'''What's Next?'''

1. 5' end additions
a. Using PCR
b. Cut at the restriction sites EcoR1 and either Bam H1 or Nco1
c. Cut plasmid and reporter with same restriction enzymes as above
d. Run a gel and purify "keeper" DNA (Ligate transform, screen, sequence)
e. Then, we take the "keeper" DNA and combine it with the insert from part

We can have steps d and e ready in advance.

2. tRNAs
a. Assemble the oligos into genes (Requires lots of calculations beforehand, but the actual steps )
b. Plasmids cut up with EcoR1 and Pst1 '''WE CAN USE THE "WASTE" PLASMID FROM THE RESTRICTION ENZYME DIGEST'''
c. Gel purify the plasmids (Ligate transform, screen, sequence)

The following tables include Master Mix measurements, agarose gel concentrations and buffers.
http://gcat.davidson.edu/GcatWiki/images/0/08/Shamitapic3.png
http://gcat.davidson.edu/GcatWiki/images/d/d1/Shamitapic4.png
http://gcat.davidson.edu/GcatWiki/images/6/6f/Shamitapic5.png

We made the Master Mix with our DNA and put it in the incubator at 10:40 AM.

'''Preparing the Gels'''

Now, we will be making 2 gels, 1 at 0.8% and 2.5% (weight to volume percent). The gel volume is 60mL.

'''Definition of 1% solution:
1 gram of agarose/ 100mL buffer'''

So for a 0.8% solution: 0.8 grams agarose/100mL buffer AND we need 60mL (fixed volume for casts run) so multiply that by 0.6.
Final concentration:

.48 grams agarose/60mL buffer

1 uL of EtBr stock for 60mL of gel.
EtBr is in the buffer so it is constantly replenishing itself even though it is moving toward one end in order to make bands on the other end visible.

After Dr. Campbell's demonstration, we each helped prepare a 0.8% buffer solution using the agarose powder, buffer and EtBr. We mixed together the first two substances in a 200mL Erlenmeyer flask and heated it for 120 seconds in the microwave. We then added EtBr and poured the gel solution into the gel cast.

'''Agar Plate'''

After lunch, we grew some more cells for each of the 11 parts in an agar plate labeled "LBAMP-AMC-9 June" so that so would have extra cells that could be stored for up to 2 weeks.
We made small circles on the agar plate and numbered them 1-11. The numbers corresponded to the following parts. We put the plate in the incubator overnight to let the cells grow.

1. B0030 AMC
2. K091111
3. S03710
4. I715039 OEH
5. S03511 ORH
6. I715039 AST
7. B0030 SDP
8. K091112
9. J31007 LJH
10. J31007 REC
11. S03511

'''Unloading the Gels'''

The following shows which parts we entered in which gels:

[[Image: screen-capture-2.png]]

We unloaded the 2.5% agarose first and after observing the bands under a UV light, put the gel into the BioRad machine to observe the bands. The image below is of a Molecular Weight Marker that shows the number of bp in control fragments of DNA according to the distance they travel down the gel.

[[Image: screen-capture-1.png]]

We obtained the following printout for the first gel that was 2.5% Agarose.
[[Image: 2009-06-09_14hr_44min.jpg]]

The bands in the first well at the bottom are those of the molecular weight marker. We skipped the lane above (to the right) of the MWM and entered the rest of the inserts as described in the above table. A light band is visible for each of the 4 other wells. In Lanes 3 and 4 we concluded we are supposed to have approximately 45 bp because the insert is 15bp + 30 bp on either side of the insert (Xba1 and Spe1 restrictions sites + sticky ends + extra bases). We used the same logic for Lanes 5 and 6 which are supposed to contain approximately 86 base pairs.

One discrepancy in this gel, however, is that Lanes 5 and 6, which according to their labels respectively contain the LacIQ promoter and the Lac IQ1 promoter. According to the information online at the parts registry website, pLacIQ1 should be the same length as pLacIQ. The gels do not show this, but instead show that the sample in Lane 5 is smaller than that in Lane 6 because Lane 5 traveled further. Leland Taylor and Romina Clemente sent out an email to Pallavi Penumetcha to ask about this because she had written a paper about this.

We then unloaded the 0.8% agarose gel and then placed this in the BioRad machine under the same protocol as above. The following image is the 0.8% Agarose.

[[Image:2009-06-09_15hr_21min.jpg]]

We observed that the bands in Lane 1 (which is now at the top) are examples of high intensity bands. The plasmids contained in the original well are twice the intensity of the insert.

In Lane 5, however, we observed several different bands. This could be due to the extra DNA that was sitting on the top of the well. We decided to re-expose the gel to get better observations on Lanes 3 and 4 because the bands were not as intense. We anticipated seeing a band at about 250 bp because this was the size of the insert (plus extra bp). After re-exposing the gel, we did not find any difference and still did not see any bands in these lanes. We have decided to redo the gel on the part S03511 and so we prepared the master mix with that DNA and incubated it so that we could run the gel tomorrow.

We also cut out the "empty" plasmids from the gel so that we could use this as vectors for the coding sequence of the suppressor tRNAs.

Tomorrow we will be planning out specific tasks for people once the oligos arrive.

==Wednesday, June 10, 2009==

We began this morning by making the agarose gel with the same protocol as yesterday. This time, since we are just re-doing both S03511 inserts, we will have a 1.8% agarose gel.

'''OUTLINE: WHERE DO WE STAND?'''

Yesterday
a. Isolated empty plasmids
b. Verified most inserts
c. Waiting for:
>tRNA oligos
>Primers
a. Will amplify the reporter gene in the plasmid itself

Next?
a. When Primers Arrive
> Amplify the reporter genes with primers (PCR takes ~3 hours)
> Calculate how much reporter plasmid we will need (Start with 50x more than we actually will need)
> Cut amplified sequence (EcoR1 and BamH1/Nco1) then purify what we need
> Cut reporter plasmid (EcoR1 and BamH1/Nco1) then purify what we need
> Ligate amplified sequence and reporter plasmid
b. When tRNA oligos arrive
> Calculate how much of each oligo we will need (takes a while...)
> "Boil and cool" tRNA oligos together
> Ligate fragments (dsDNA) into plasmids we isolated yesterday (~5 minutes)
> Transform the plasmids (~20 minutes)

* LOOK THROUGH LIGATION PROTOCOL (How to assemble genes)
* LOOK THROUGH TRANSFORMATION PROTOCOL

''What we need to get done today'':
1. Prepare the control for the Tet Resistance by BioBricking the part pLac+RBS (S03511) with the TetA (J31007).
2. Check the part number for RFP and also for pLac+RBS+RFP
3. Read through ligation protocol
4. See how many nanograms of plasmid we will need for the tRNAs and for the inserts and multiply this number by 50.
> Check if we have enough plasmids from our mini-prep
5. Purify the plasmids from the gel

After we made this outline, Leland, Olivia and I analyzed the gel we ran this morning and did not see a more than one band in the 2 lanes that contained DNA from S03511 (image below). Since we did not see multiple bands in yesterday's gel either, we conclude that there is something wrong with the DNA rather than with the restriction enzymes that we used to cut it. We concluded that there is probably not an insert in this plasmid both the part and the plasmid are useless.

[[Image: 2009-06-10 10hr 30min.jpg]]

Now, we do not have an immediately accessible promoter. We have decided to order the pBAD promoter from Missouri Western and we will attach the RBS+Tet to this plasmid. Our insert for this will be the larger gene because it is easier to manipulate in the gel than the small gene.

Alyndria Thompson and Romina Clemente are figuring out the size of the plasmids including the reporter genes.

After lunch we performed several steps to isolate and purify the gel to get the empty plasmids will be the vectors for the tRNA DNA. Then, we used the NanoDrop to assess the purity of our DNA. We found that the plasmids were more pure than we had expected. The following values were obtained:

Abs 1.850
A-260 10 mm path 0.172
A-280 10 mm path 0.075
260/280 2.28
260/230 0.09

We then followed the protocol for preparing the agar plates. We put the plate with the agar solution in the Autoclave.

While the solution was in the autoclave, we discussed that we don't really need a promoter in front of RBS+Tet for the gene to be expressed because Tet expression is so strong that the gene would translate without a promoter. However, it would be a good idea to have the promoter because if it was possible to reinforce the translation of the reporter protein, we wanted to do that.

We found that there were some tubes in the lab fridge that contained the pBAD and pLac promoters with RBS. We decided to use these as the promoter and RBS for our experiments. The pLac promoter will be used for RFP and the pBad promoter used with Tet Resistance. We decided to use the pBad with Tet because it is weaker in comparison to pLac. As expression of the Tet gene can be toxic to a cell, we do not want to overexpress the gene with a stronger promoter.

Once the agar solution was sterilized in the Autoclave, we removed it, cooled it, and pipetted the antibiotic resistance into it. We then poured the solution into the plates, removed any bubbles, and let the gel form.

Alyndria Thompson and Romina Clemente concluded that we had more than enough recieving vectors for both the RFP and Tetracycline genes. Since we need 50 nanograms, it is wise to have at least 50 times that much that we can work with, so we need at least 2,500 ng.

RFP 1: 77.6 x 4 = 310.4 ng/uL
x 20 uL = 3,272 ng
RFP 2: 59.5 x 4 = 238 ng/uL
x 20 uL = 4,760 ng
Tet: 40.9 x 4 = 163.6 ng/uL
x 20 uL = 3,272 ng

According to these calculations we have more than 50 times what we need for both instances of RFP and Tet.

Tomorrow, we can prepare the Master Mix for the digestions of both reporter plamids and then add the restriction enzymes once they arrive. Then, we can follow through with the rest of the plans we had made above in the section titles "Next?".

==Thursday, June 11, 2009==

We began this morning by calculating how many uL we would need for each of the mixes for digesting our reporter plasmids. We decided to double the final volume of each mix to 40uL instead of 20uL. Since we knew the concentration of each plasmid and also knew we needed 2500 ng, we cross multiplied to find the uL of DNA we would need for each.

Concentration Calculations:
310.4 ng/1 uL = 2500ng/x uL
x = 8.05 uL

Here is a chart indicating how much of each we used for the 2 mixes.

[[Image: DoubledigestRFPTetR.png]]

On the Promega website, we found a list of compatible buffers for double digestions. Buffers E and H are compatible for the Tet plasmid and RFP plasmid digestions respectively. We fortunately have both buffers in stock.

[[Image:BamEcoR1.png]] [[Image:NcoEcoR1.png]]

We waited for our waited for the restriction enzymes to arrive and in the meantime, we transformed the plasmids with pLac+RBS, pBad, and pBad+RBS into ''E.coli'' cells. We want the ''E.coli'' cells to replicate the plasmids with these genes because we have a limited amount of genes. [http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_Transformation.html Zippy Plasmid Transformation]

Once our oligos and restriction enzymes arrived, we got to work right away!

'''PCR (Polymerase Chain Reaction)'''

We calculated how much plasmid we will need for the PCR. We decided to dilute the concentration of our plasmids a hundred fold so that we could get pipetable amounts.

''Change in Concentrations''
310.4 ng/uL x 1/100 (dilution factor) = 3.104 ng/uL (RFP)
163.6 ng/uL x 1/100 (dilution factor) = 0.164 ng/uL (Tet)

''Example shown with RFP''
3.104 ng/1 uL = 1ng/ x uL, x = .32
For Tet, x = .61

We also calculated how much of each primer we would need with the following equation:

C1V1 = C2V2
(100 uM) (x) = (1 uM)( 100 uL)
x = 1 uL

[[Image: screen-capture-8.png]]

Each of the 5 of us made had 2 tubes (one for each reporter protein) because there were 5 different forward primers.To each tube, we added the template DNA (either RFP or Tet), water, the respective reverse primer (either RFP or Tet), and the forward primer depending on which 5mer we had chosen. My 5mer was CCAUC. Finally, we added the Master Mix last because this contained the enzyme (DNA polymerase) that would amplify our desired sequence.

[[Image: Pcrtubes.png]]

We followed the temperature cycles so that PCR could occur. The annealing temperature needed to be 5C less than the smallest melting temperature of the primers. Since the reverse primer of RFP had a melting temp of 66C, we set the temperature of the 3rd cycle on 61C.

[[Image: Temperaturecycle.png]]

'''tRNA Oligo Assembly'''

We needed to calculate the concentration of each of the oligo before carrying out the assembly. The stock concentration of each of the oligo was 100 uM. Since we wanted to have 5uM of the oligo in the test tube, and we know that final volume of the test tube is 20 uL, we just cross multiplied to find the amount we needed to add to the tubes to get this concentration.

(100uM)x = (5uM)(20uL)
x = 1 uL

We added 1 uL of each oligo to the tubes.

We had six total tRNAs we needed to assemble so each of us took one FML (one person took two) and to each tube we added 1uL of each oligo for a total of 7uL, 2uL of buffer, and 11uL of water for a total volume of 20 uL. Five of the oligos were constant and 2 varied for each of the different tRNAs. So everyone pipetted the same 5 oligos and everyone also had 2 unique oligos to add. When all of the oligos were added and the tubes ready, we boiled the tubes at 50C for 5 minutes. We then allowed the tubes to remain in the water to cool very slowly overnight so that the annealing process could take place.

The last thing we did at the end of the day was finish off our plasmid digestion that we had begun in the morning. We added the buffer, DNA, water, and restriction enzymes and placed this in the incubator for overnight digestion.

'''To Do''':

1. Isolate our desired dsDNA from the PCR.
2. Verify our tRNA inserts and eventually transform these into plasmids.
3. Check our agar plates to see colonies that have replicated our pLac+RBS, pBAD+RBS, and pBAD genes.

==Friday, June 12, 2009==

This morning, we began by outlining the main items on today's agenda.

1. tRNAs: Ligation, Transformation
2. Digestion reporters (run all of it and gel purify)
3. Transformed cells
4. PCR: Gel run an aliquot to verify (5uL)

We used the equation given by the Ligation protocol to calculate how many nanograms of insert we would need.

[[Insert: screen-capture-2.png]]

X nanograms of insert = [(2)(144)(50 ng linearalized plasmid)]/2038
X = 7.0657 ng of insert

We calculated our initial concentration in ng/uL of insert as shown below.

[[Image: screen-capture-1.png]]

IGEM Notebook

2009-06-12T22:22:37Z

ShPunjabi: /* Friday, June 12, 2009 */

==Wednesday, June 3, 2009==

Romina Clemente and I are trying to find suitable reporter proteins to use. Yesterday, Leland Taylor and Alyndria Thompson were working on ways to insert the gene sequences into the plasmid. Upon seeing how they wanted to manipulated the reporter gene to include the logical clauses, we came up with a few criteria for the reporter genes we would use. The following criteria for genes are listed in order of the broadest aspect to look at to the narrowest aspect:
a) Doesn't contain restriction sites for the 4 restriction enzymes (EcoR1, Xbal, Spel, Pst1) used to cleave the Biobrick part out of the plasmid.
b) Contains 6 cutter restriction sites.
c) These restriction enzymes aren't blunt (cleave straight down at one spot).
d) These restriction sites are close to thge 5' (beginning) end of the sequence.
e) These enzymes are easiest to work with and cheapest.
We are finding the part numbers of the reporter genes we want to use (antibiotic resistance, fluorescence, LacZ) through our own GCAT because we know these ones work. We are then locating these parts on the parts registry [1] website. We copied and pasted the gene sequences we obtained from the registry onto the ApE software [2]. From here, we were able to generate a genetic map of each gene that outlined each restriction site that fit our criteria. We put each genetic map, alongside the part number used, into a Word document.

http://gcat.davidson.edu/GcatWiki/images/0/0e/Restriction_Site_Mapping_on_Reporter_Genes.doc

Later on in the day, we decided that the best way to test for suppression would involve placing the 5mers at the beginning of the reporter gene instead of inserting them into the reporter gene. To do this, we decided that the gene along with its start codon need to be expressed after the 5mer. We will insert a BioBrick plasmid with a 5mer and start codon that will be incorporated before the gene:
RBS-6-8nt-ATG-5mer--ATG-gene

Leland Taylor assembled the oligos that we will need for the BioBrick pieces.

In order to test whether the reporter protein would be expressed regardless of the 5mer being suppressed or not, we would need to remove the suppressor tRNAs from the cell.

Meanwhile, I helped figure out the coding sequence that we will need for the suppressor tRNAs that will bind to the 5mers. We decided that we would be using the following suppressor tRNAs on the Davidson side: CUAGU, CCCUC, CGGUC, CCAUC, and CCACU. We searched through several papers, with little result and finally emailed Dr. Christopher Anderson of UC Berkeley to request the DNA sequence. He emailed us back with a generic DNA code that we could use to create the tRNA; however, for each suppressor tRNA we would use, we would need to change base pairs in the anticodon loop.

The length of the tRNA is 92 nt. The anticodon loop is comprised of 9 nt. Once we have BioBricked the DNA sequence with 4 restriction sites and supplemental nucleotides, the length of the entire gene is 144 nucleotides. This is made up of the 92 nt +22 nt on each side of the sequence (44) + 4 nt on each side of the restriction ends (8).

At the end of the day, we decided to use tetracycline resistance and RFP as our reporter proteins for this tester experiment.

==Thursday, June 4, 2009==

This morning, I began the day by learning how to use the PEARL prgram. Olivia Ho-Shing sat with me and walked me through the program script. And then, Leland Taylor and Shashank Suresh had a problem they wanted us to solve. Leland explained that we need to make sure there is a stop codon in the gene if we fail to suppress the 5mer. The stop codon needs to be before we find another 5mer. They also wanted us to run through both the RFP and Tet Resistance genes to make sure that they did not contain any of the anticodon sequences from our 5 frameshift suppressor tRNAs so as to throw the translation out of frame. They further wanted us to confirm that if we do have successful suppression, there are no stop codons in the middle of the RFP and Tet Resistance genes. PEARL did not find any matches for the suppressor tRNAs within genes. We found 3 matches for stop codons UAA. Two of these occured at the end of the Tet and one at the end of RFP, where they should normally be to end translation into a protein.

Following this, Olivia Ho-Shing and I tried to determine how we can add BioBrick ends to our single stranded DNA sequences that code for the suppressor tRNAs. Our ultimate goal is to find complements for these single strands so they can be put into plasmids. We found the standard prefixes and suffixes for sequences that do not contain ATG (becuase this sequence is a functional RNA and will not be translated) and we placed these before and after the altered 92bp sequence that Dr. Anderson provided us with. The prefixes and suffixes are necessary because they are the extra nucleotides that will be mimicking a "restriction enzyme cutting site". The single strand appeared as below after the Bio Brick prefixes and suffixes were added:

22ntPREFIX--32nt--VARIABLE ANTICODON LOOP--51nt--21ntSUFFIX

We used the suppressor codon CUAGU as our example and placed its appropriate anticodon loop in the sequence. We put this into the lancelator that would give us the other strand as well as the oligos we would need for ideal construction of this double stranded DNA fragment to put into the plasmid. We recieved a total of 4 oligos, 2 of which were variable and 2 of which were constant for all 6 different suppressor tRNAs. Using this double strand, we simulated a restriction enzyme digestion by EcoRI and PstI. We took off the G at both ends of the top strand and then removed the first five nucleotides on each end of the bottom strand. This made the top strand longer than the bottom one. We then proceeded to take the two variable strands and changed the anticodon loop nt for each suppressor tRNA we used.

Around lunchtime, we encountered a problem with the BioBrick scar in the "ATG-5mer subpiece" of the test project. It turns out that after annealing the sticky ends of the Xba1 and Spe1 sites, the scar created had a stop codon TAG (UAG) that was in frame with the suppressed codon. When the Xba1 site on insert anneals to the Spe1 site on the plasmid (the green portions), we get a scar that reads TACTAG.
[[Image:screen-capture.png]]

For the afternoon, Olivia Ho-Shing and I finished assembly of the oligos of the suppressor tRNA codes. We assembled the document below:

http://gcat.davidson.edu/GcatWiki/images/5/5e/TRNAoligostoOrder.doc

We were assigned tasks of creating "cartoons" to illustrate the phenomenon that had occurred. No one could successfully do this however, because we could not understand how the piece had formed. The parts registry website provided wrong information on the restriction site of Spe1 which slowed our efficiency. This project turned into an effort to try to find two other different restriction enzymes that have complementary sticky ends other than Xba1 and Spe1. We want to get rid oc the scar that contains TACTAG.

We ended up deciding that a hybrid of two ideas was needed: restriction enzymes would need to be used to make several reporter gene sequences with different beginnings dependending on the suppressor tRNA sequence needed.

==Friday, June 5, 2009==

Our goal for this morning was to discuss the hybrid PCR approach to assemble the part that comes before the gene we wanted to express. We decided that we would have a BioBrick prefix and a 5mer followed by a sequence of nucleotides whose identity was up to us. Using PCR we would get the second double strand if we have the following:

BioBrick Prefix--ATG--5mer--gene until we get to restriction site

The PCR primer would attach to the ATG and the 5mer and would replicate the complementary base pairs to make a double stranded DNA sequence. As several of us were confused about PCR methods, we looked at the online link provided by Alyndria Thompson's link to the PCR interactive website which was very helpful. We also did some research to discover that the restriction enzyme BseRI (which is contained in the YFP that we wanted to alter) is not compatible with any other restriction enzyme. This is good because we did not want to plasmid containing this gene to close up with Xba1 once we digested it.

The team experienced some confusion with the hybrid idea and spoke to Clif Davis of Missouri Western University on the phone. He told us that there will be one part that is a fragment of the beginning of some nucleotides as a restriction cleaving site, ATG, the logical clause, and the start of the YFP gene that will be placed into a plasmid containing a space. To the left of the space will be the BioBrick Prefix and the right of the space will contain the rest of the genes in the YFP. Therefore, we decided not to alter any BioBrick ends and just look at restriction sites at the beginning of reporter proteins. The small fragment that will be placed into the plasmid will be made by PCR. The PCR will attach a primer onto the ATG and 5mer and replicate.

While we were discussing this, we expanded upon the idea that we can do whatever we want with the "suffix" portion between the 5mer and the beginning of YFP. We decided it would be best to remove ATG and add the first 5 amino acids and the restriction enzyme site. Along with this, if we have the 5mer coding for a certain amino acid (ie methionine) we could replace another codon that codes for methionine WITH that 5mer so long as this codon is contained within the first 15 nt. This way, we would not be altering the protein.

Olivia Ho-Shing came up with an idea of cascading PCR effect for longer logical clauses. Post translational modification of the protein was another one of her ideas. This applies because although we may code for the protein in a given way, the protein may modify or fix itself up to perform a slightly different function in its tertiary form.

Dr. Campbell suggested that we can look up restriction enzymes 400-500 base pairs into the reporter protein we would like to use; however, we have more flexibility in our procedure if we choose more "hardy" cutters. So instead, we could cut with EcoRI instead of with Xba1.

During the afternoon, we set about three tasks:
1) Which primers to use
2) Which promoters to use
3) Which restriction enzymes and how they will determine use of reporter proteins

Olivia Ho-Shing and I began looking up which restriction enzymes were strong so we could base our reporters on that. We began with Tet and RFP since those were the two reporters we had started out with choosing. I found a website that provided enzyme cleavage efficiency based upon the proximity of the site to the end of the PCR fragment that contains it. We discovered the following based upon the attached documents:

http://gcat.davidson.edu/GcatWiki/images/8/8f/Restriction_Enzyme_Cleavage_Efficiency.doc
http://gcat.davidson.edu/GcatWiki/images/9/9a/07Jun09_RE_Cutting_Efficiency_oh.doc

We decided that these two reporters contained efficient enzymes in a good quantity to choose from. On Monday, we will look through these enzymes once more and finalize which enzymes we would like to use with RFP and Tet.

==Sunday, June 7, 2009==

We did preparations for the mini prep this evening. The following notes are courtesy of Olivia Ho-Shing.

1. Sterically transfer ~2mL LB+AMP (AMP are ampicillin resistant plasmids) *Never put fluid back into bottle once it has entered pipette*
2. Use forceps to grab toothpick, scrap some frozen cells, rub in tube with top

'''Tubes'''

S03511 (2)

K091111

B0030

I715039

K091112

I715039

J31007 (2)

B0034

E1010

S03710

==Monday, June 8, 2009==

We reviewed the PCR process this morning as well as the restriction enzyme cleavage process.

Then, Leland Taylor, Olivia Ho-Shing and I all worked on converting the tRNA oligos we made to the template that Missouri Western used. The oligos we had previously made were different from the ones that Dr. Anderson had originally emailed us because further communication between Dr. Anderson and Missouri Western resulted in a better template.

We tried several times to minimize the number of oligos while keeping their individual lengths under 80bp. We managed to do this in the end by creating 7 oligos, 2 of which are variable between the suppressor tRNAs.
We reviewed the following document and finalized the oligos that we needed to order for the frameshift suppressor tRNAs. We decided to keep both the 10bp and 9bp anticodons of CCAUC.

I also looked up the amino acids that corresponded to the FS (frameshift suppressor) tRNAs for future use:

1. CUAGU = Leu
2. CCCUC = Pro
3. CGGUC = Arg
4. CCACU = Pro
5. CCAUC* = Pro
*9 and 10 bp anticodons

Also, we have decided to use NcoI as the restriction enzyme for RFP. This cleavage site occurs 419 bp into the gene. We have decided to use BamHI as the enzyme for Tetracycline Resistance and the cleavage site occurs 290bp into the gene. Both of these enzymes are very powerful and hardy.

We also looked over Missouri Western's tRNA oligos and primer oligos which we approved.
Our oligos were also approved by Missouri Western and Dr. Campbell ordered them while we supervised.

In the afternoon we did the miniprep. The mini prep directions were as appear in the link below.

http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_MiniPrep.html

We then worked with the Nano Prep to dilute our solution of DNA to an absorption of 1.5 and then we made labels for the tubes. The chart below shows relevant information we obtained from the NanoDrop for each of the reporter plasmids.

[[Image: Screen-capture-6.png]]

==Tuesday, June 9, 2009==

'''Preparations for restriction enzymes digestion of plasmids'''

Purpose: To determine the size of the plasmid inserts and ensure that plasmids are what we extracted from the cells during the mini-prep.

Things we need:
DNA ~3uL
Buffer 10x (Want to dilute to 1x) so add 2uL
Enzyme 1 ~1uL
Enzme 2 ~1uL
Water ~13uL
Final Volunter about 20 uL.

Need to choose the right buffer for the restriction enzymes. All buffers come in 10x (10 times more concentrated than what you want it to be when you use it). [http://gcat.davidson.edu/GcatWiki/index.php/Davidson_Missouri_W/Double_Digest_Guide Buffer Website]

Total volume of enzymes has to be 10% or less than final volume (most volume 2uL). Barely submerge the pipet tip into the solution when pipeting the enzymes.
Star (*) activity: Restriction enzyme stops cutting specifically and starts cutting everywhere.

DNA can't be seen by itself so we add EtBr (the bigger the insert, the more will bind) so bigger strands appear brighter. samples.

The order of entry doesn't matter, but make sure to enter the enzymes last.

Will have lots of labeled tubes and will put 3uL of appropriate plasmids into the tubes.
In the meantime, we'll make a "Master Mix" of all the other ingredients in a bigger tube. Always make enough volume for one extra tube.

Enzymes usually have an optimal temperature of 37C. We will let this reaction go for an hour and then we will run it on a gel.

'''Gel Electrophoresis Protocol'''

Website online for ideal gel concentration for resolving different sizes of molecules [http://gcat.davidson.edu/iGEM08/gelwebsite/gelwebsite.html Gel Concentration Website].
See protocol on the Davidson/Missouri Western Wookie for running molecules in the gel and how to digest with restriction enzymes.

'''What's Next?'''

1. 5' end additions
a. Using PCR
b. Cut at the restriction sites EcoR1 and either Bam H1 or Nco1
c. Cut plasmid and reporter with same restriction enzymes as above
d. Run a gel and purify "keeper" DNA (Ligate transform, screen, sequence)
e. Then, we take the "keeper" DNA and combine it with the insert from part

We can have steps d and e ready in advance.

2. tRNAs
a. Assemble the oligos into genes (Requires lots of calculations beforehand, but the actual steps )
b. Plasmids cut up with EcoR1 and Pst1 '''WE CAN USE THE "WASTE" PLASMID FROM THE RESTRICTION ENZYME DIGEST'''
c. Gel purify the plasmids (Ligate transform, screen, sequence)

The following tables include Master Mix measurements, agarose gel concentrations and buffers.
http://gcat.davidson.edu/GcatWiki/images/0/08/Shamitapic3.png
http://gcat.davidson.edu/GcatWiki/images/d/d1/Shamitapic4.png
http://gcat.davidson.edu/GcatWiki/images/6/6f/Shamitapic5.png

We made the Master Mix with our DNA and put it in the incubator at 10:40 AM.

'''Preparing the Gels'''

Now, we will be making 2 gels, 1 at 0.8% and 2.5% (weight to volume percent). The gel volume is 60mL.

'''Definition of 1% solution:
1 gram of agarose/ 100mL buffer'''

So for a 0.8% solution: 0.8 grams agarose/100mL buffer AND we need 60mL (fixed volume for casts run) so multiply that by 0.6.
Final concentration:

.48 grams agarose/60mL buffer

1 uL of EtBr stock for 60mL of gel.
EtBr is in the buffer so it is constantly replenishing itself even though it is moving toward one end in order to make bands on the other end visible.

After Dr. Campbell's demonstration, we each helped prepare a 0.8% buffer solution using the agarose powder, buffer and EtBr. We mixed together the first two substances in a 200mL Erlenmeyer flask and heated it for 120 seconds in the microwave. We then added EtBr and poured the gel solution into the gel cast.

'''Agar Plate'''

After lunch, we grew some more cells for each of the 11 parts in an agar plate labeled "LBAMP-AMC-9 June" so that so would have extra cells that could be stored for up to 2 weeks.
We made small circles on the agar plate and numbered them 1-11. The numbers corresponded to the following parts. We put the plate in the incubator overnight to let the cells grow.

1. B0030 AMC
2. K091111
3. S03710
4. I715039 OEH
5. S03511 ORH
6. I715039 AST
7. B0030 SDP
8. K091112
9. J31007 LJH
10. J31007 REC
11. S03511

'''Unloading the Gels'''

The following shows which parts we entered in which gels:

[[Image: screen-capture-2.png]]

We unloaded the 2.5% agarose first and after observing the bands under a UV light, put the gel into the BioRad machine to observe the bands. The image below is of a Molecular Weight Marker that shows the number of bp in control fragments of DNA according to the distance they travel down the gel.

[[Image: screen-capture-1.png]]

We obtained the following printout for the first gel that was 2.5% Agarose.
[[Image: 2009-06-09_14hr_44min.jpg]]

The bands in the first well at the bottom are those of the molecular weight marker. We skipped the lane above (to the right) of the MWM and entered the rest of the inserts as described in the above table. A light band is visible for each of the 4 other wells. In Lanes 3 and 4 we concluded we are supposed to have approximately 45 bp because the insert is 15bp + 30 bp on either side of the insert (Xba1 and Spe1 restrictions sites + sticky ends + extra bases). We used the same logic for Lanes 5 and 6 which are supposed to contain approximately 86 base pairs.

One discrepancy in this gel, however, is that Lanes 5 and 6, which according to their labels respectively contain the LacIQ promoter and the Lac IQ1 promoter. According to the information online at the parts registry website, pLacIQ1 should be the same length as pLacIQ. The gels do not show this, but instead show that the sample in Lane 5 is smaller than that in Lane 6 because Lane 5 traveled further. Leland Taylor and Romina Clemente sent out an email to Pallavi Penumetcha to ask about this because she had written a paper about this.

We then unloaded the 0.8% agarose gel and then placed this in the BioRad machine under the same protocol as above. The following image is the 0.8% Agarose.

[[Image:2009-06-09_15hr_21min.jpg]]

We observed that the bands in Lane 1 (which is now at the top) are examples of high intensity bands. The plasmids contained in the original well are twice the intensity of the insert.

In Lane 5, however, we observed several different bands. This could be due to the extra DNA that was sitting on the top of the well. We decided to re-expose the gel to get better observations on Lanes 3 and 4 because the bands were not as intense. We anticipated seeing a band at about 250 bp because this was the size of the insert (plus extra bp). After re-exposing the gel, we did not find any difference and still did not see any bands in these lanes. We have decided to redo the gel on the part S03511 and so we prepared the master mix with that DNA and incubated it so that we could run the gel tomorrow.

We also cut out the "empty" plasmids from the gel so that we could use this as vectors for the coding sequence of the suppressor tRNAs.

Tomorrow we will be planning out specific tasks for people once the oligos arrive.

==Wednesday, June 10, 2009==

We began this morning by making the agarose gel with the same protocol as yesterday. This time, since we are just re-doing both S03511 inserts, we will have a 1.8% agarose gel.

'''OUTLINE: WHERE DO WE STAND?'''

Yesterday
a. Isolated empty plasmids
b. Verified most inserts
c. Waiting for:
>tRNA oligos
>Primers
a. Will amplify the reporter gene in the plasmid itself

Next?
a. When Primers Arrive
> Amplify the reporter genes with primers (PCR takes ~3 hours)
> Calculate how much reporter plasmid we will need (Start with 50x more than we actually will need)
> Cut amplified sequence (EcoR1 and BamH1/Nco1) then purify what we need
> Cut reporter plasmid (EcoR1 and BamH1/Nco1) then purify what we need
> Ligate amplified sequence and reporter plasmid
b. When tRNA oligos arrive
> Calculate how much of each oligo we will need (takes a while...)
> "Boil and cool" tRNA oligos together
> Ligate fragments (dsDNA) into plasmids we isolated yesterday (~5 minutes)
> Transform the plasmids (~20 minutes)

* LOOK THROUGH LIGATION PROTOCOL (How to assemble genes)
* LOOK THROUGH TRANSFORMATION PROTOCOL

''What we need to get done today'':
1. Prepare the control for the Tet Resistance by BioBricking the part pLac+RBS (S03511) with the TetA (J31007).
2. Check the part number for RFP and also for pLac+RBS+RFP
3. Read through ligation protocol
4. See how many nanograms of plasmid we will need for the tRNAs and for the inserts and multiply this number by 50.
> Check if we have enough plasmids from our mini-prep
5. Purify the plasmids from the gel

After we made this outline, Leland, Olivia and I analyzed the gel we ran this morning and did not see a more than one band in the 2 lanes that contained DNA from S03511 (image below). Since we did not see multiple bands in yesterday's gel either, we conclude that there is something wrong with the DNA rather than with the restriction enzymes that we used to cut it. We concluded that there is probably not an insert in this plasmid both the part and the plasmid are useless.

[[Image: 2009-06-10 10hr 30min.jpg]]

Now, we do not have an immediately accessible promoter. We have decided to order the pBAD promoter from Missouri Western and we will attach the RBS+Tet to this plasmid. Our insert for this will be the larger gene because it is easier to manipulate in the gel than the small gene.

Alyndria Thompson and Romina Clemente are figuring out the size of the plasmids including the reporter genes.

After lunch we performed several steps to isolate and purify the gel to get the empty plasmids will be the vectors for the tRNA DNA. Then, we used the NanoDrop to assess the purity of our DNA. We found that the plasmids were more pure than we had expected. The following values were obtained:

Abs 1.850
A-260 10 mm path 0.172
A-280 10 mm path 0.075
260/280 2.28
260/230 0.09

We then followed the protocol for preparing the agar plates. We put the plate with the agar solution in the Autoclave.

While the solution was in the autoclave, we discussed that we don't really need a promoter in front of RBS+Tet for the gene to be expressed because Tet expression is so strong that the gene would translate without a promoter. However, it would be a good idea to have the promoter because if it was possible to reinforce the translation of the reporter protein, we wanted to do that.

We found that there were some tubes in the lab fridge that contained the pBAD and pLac promoters with RBS. We decided to use these as the promoter and RBS for our experiments. The pLac promoter will be used for RFP and the pBad promoter used with Tet Resistance. We decided to use the pBad with Tet because it is weaker in comparison to pLac. As expression of the Tet gene can be toxic to a cell, we do not want to overexpress the gene with a stronger promoter.

Once the agar solution was sterilized in the Autoclave, we removed it, cooled it, and pipetted the antibiotic resistance into it. We then poured the solution into the plates, removed any bubbles, and let the gel form.

Alyndria Thompson and Romina Clemente concluded that we had more than enough recieving vectors for both the RFP and Tetracycline genes. Since we need 50 nanograms, it is wise to have at least 50 times that much that we can work with, so we need at least 2,500 ng.

RFP 1: 77.6 x 4 = 310.4 ng/uL
x 20 uL = 3,272 ng
RFP 2: 59.5 x 4 = 238 ng/uL
x 20 uL = 4,760 ng
Tet: 40.9 x 4 = 163.6 ng/uL
x 20 uL = 3,272 ng

According to these calculations we have more than 50 times what we need for both instances of RFP and Tet.

Tomorrow, we can prepare the Master Mix for the digestions of both reporter plamids and then add the restriction enzymes once they arrive. Then, we can follow through with the rest of the plans we had made above in the section titles "Next?".

==Thursday, June 11, 2009==

We began this morning by calculating how many uL we would need for each of the mixes for digesting our reporter plasmids. We decided to double the final volume of each mix to 40uL instead of 20uL. Since we knew the concentration of each plasmid and also knew we needed 2500 ng, we cross multiplied to find the uL of DNA we would need for each.

Concentration Calculations:
310.4 ng/1 uL = 2500ng/x uL
x = 8.05 uL

Here is a chart indicating how much of each we used for the 2 mixes.

[[Image: DoubledigestRFPTetR.png]]

On the Promega website, we found a list of compatible buffers for double digestions. Buffers E and H are compatible for the Tet plasmid and RFP plasmid digestions respectively. We fortunately have both buffers in stock.

[[Image:BamEcoR1.png]] [[Image:NcoEcoR1.png]]

We waited for our waited for the restriction enzymes to arrive and in the meantime, we transformed the plasmids with pLac+RBS, pBad, and pBad+RBS into ''E.coli'' cells. We want the ''E.coli'' cells to replicate the plasmids with these genes because we have a limited amount of genes. [http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_Transformation.html Zippy Plasmid Transformation]

Once our oligos and restriction enzymes arrived, we got to work right away!

'''PCR (Polymerase Chain Reaction)'''

We calculated how much plasmid we will need for the PCR. We decided to dilute the concentration of our plasmids a hundred fold so that we could get pipetable amounts.

''Change in Concentrations''
310.4 ng/uL x 1/100 (dilution factor) = 3.104 ng/uL (RFP)
163.6 ng/uL x 1/100 (dilution factor) = 0.164 ng/uL (Tet)

''Example shown with RFP''
3.104 ng/1 uL = 1ng/ x uL, x = .32
For Tet, x = .61

We also calculated how much of each primer we would need with the following equation:

C1V1 = C2V2
(100 uM) (x) = (1 uM)( 100 uL)
x = 1 uL

[[Image: screen-capture-8.png]]

Each of the 5 of us made had 2 tubes (one for each reporter protein) because there were 5 different forward primers.To each tube, we added the template DNA (either RFP or Tet), water, the respective reverse primer (either RFP or Tet), and the forward primer depending on which 5mer we had chosen. My 5mer was CCAUC. Finally, we added the Master Mix last because this contained the enzyme (DNA polymerase) that would amplify our desired sequence.

[[Image: Pcrtubes.png]]

We followed the temperature cycles so that PCR could occur. The annealing temperature needed to be 5C less than the smallest melting temperature of the primers. Since the reverse primer of RFP had a melting temp of 66C, we set the temperature of the 3rd cycle on 61C.

[[Image: Temperaturecycle.png]]

'''tRNA Oligo Assembly'''

We needed to calculate the concentration of each of the oligo before carrying out the assembly. The stock concentration of each of the oligo was 100 uM. Since we wanted to have 5uM of the oligo in the test tube, and we know that final volume of the test tube is 20 uL, we just cross multiplied to find the amount we needed to add to the tubes to get this concentration.

(100uM)x = (5uM)(20uL)
x = 1 uL

We added 1 uL of each oligo to the tubes.

We had six total tRNAs we needed to assemble so each of us took one FML (one person took two) and to each tube we added 1uL of each oligo for a total of 7uL, 2uL of buffer, and 11uL of water for a total volume of 20 uL. Five of the oligos were constant and 2 varied for each of the different tRNAs. So everyone pipetted the same 5 oligos and everyone also had 2 unique oligos to add. When all of the oligos were added and the tubes ready, we boiled the tubes at 50C for 5 minutes. We then allowed the tubes to remain in the water to cool very slowly overnight so that the annealing process could take place.

The last thing we did at the end of the day was finish off our plasmid digestion that we had begun in the morning. We added the buffer, DNA, water, and restriction enzymes and placed this in the incubator for overnight digestion.

'''To Do''':

1. Isolate our desired dsDNA from the PCR.
2. Verify our tRNA inserts and eventually transform these into plasmids.
3. Check our agar plates to see colonies that have replicated our pLac+RBS, pBAD+RBS, and pBAD genes.

==Friday, June 12, 2009==

This morning, we began by outlining the main items on today's agenda.

1. tRNAs: Ligation, Transformation
2. Digestion reporters (run all of it and gel purify)
3. Transformed cells
4. PCR: Gel run an aliquot to verify (5uL)

We used the equation given by the Ligation protocol to calculate how many nanograms of insert we would need.

[[Insert: screen-capture-2.png]]

X nanograms of insert = [(2)(144)(50 ng linearalized plasmid)]/2038
X = 7.0657 ng of insert

We calculated our initial concentration in ng/uL of insert as shown below.

[[Image: screen-capture-2.png]]

IGEM Notebook

2009-06-12T16:30:23Z

ShPunjabi: /* Thursday, June 11, 2009 */

==Wednesday, June 3, 2009==

Romina Clemente and I are trying to find suitable reporter proteins to use. Yesterday, Leland Taylor and Alyndria Thompson were working on ways to insert the gene sequences into the plasmid. Upon seeing how they wanted to manipulated the reporter gene to include the logical clauses, we came up with a few criteria for the reporter genes we would use. The following criteria for genes are listed in order of the broadest aspect to look at to the narrowest aspect:
a) Doesn't contain restriction sites for the 4 restriction enzymes (EcoR1, Xbal, Spel, Pst1) used to cleave the Biobrick part out of the plasmid.
b) Contains 6 cutter restriction sites.
c) These restriction enzymes aren't blunt (cleave straight down at one spot).
d) These restriction sites are close to thge 5' (beginning) end of the sequence.
e) These enzymes are easiest to work with and cheapest.
We are finding the part numbers of the reporter genes we want to use (antibiotic resistance, fluorescence, LacZ) through our own GCAT because we know these ones work. We are then locating these parts on the parts registry [1] website. We copied and pasted the gene sequences we obtained from the registry onto the ApE software [2]. From here, we were able to generate a genetic map of each gene that outlined each restriction site that fit our criteria. We put each genetic map, alongside the part number used, into a Word document.

http://gcat.davidson.edu/GcatWiki/images/0/0e/Restriction_Site_Mapping_on_Reporter_Genes.doc

Later on in the day, we decided that the best way to test for suppression would involve placing the 5mers at the beginning of the reporter gene instead of inserting them into the reporter gene. To do this, we decided that the gene along with its start codon need to be expressed after the 5mer. We will insert a BioBrick plasmid with a 5mer and start codon that will be incorporated before the gene:
RBS-6-8nt-ATG-5mer--ATG-gene

Leland Taylor assembled the oligos that we will need for the BioBrick pieces.

In order to test whether the reporter protein would be expressed regardless of the 5mer being suppressed or not, we would need to remove the suppressor tRNAs from the cell.

Meanwhile, I helped figure out the coding sequence that we will need for the suppressor tRNAs that will bind to the 5mers. We decided that we would be using the following suppressor tRNAs on the Davidson side: CUAGU, CCCUC, CGGUC, CCAUC, and CCACU. We searched through several papers, with little result and finally emailed Dr. Christopher Anderson of UC Berkeley to request the DNA sequence. He emailed us back with a generic DNA code that we could use to create the tRNA; however, for each suppressor tRNA we would use, we would need to change base pairs in the anticodon loop.

The length of the tRNA is 92 nt. The anticodon loop is comprised of 9 nt. Once we have BioBricked the DNA sequence with 4 restriction sites and supplemental nucleotides, the length of the entire gene is 144 nucleotides. This is made up of the 92 nt +22 nt on each side of the sequence (44) + 4 nt on each side of the restriction ends (8).

At the end of the day, we decided to use tetracycline resistance and RFP as our reporter proteins for this tester experiment.

==Thursday, June 4, 2009==

This morning, I began the day by learning how to use the PEARL prgram. Olivia Ho-Shing sat with me and walked me through the program script. And then, Leland Taylor and Shashank Suresh had a problem they wanted us to solve. Leland explained that we need to make sure there is a stop codon in the gene if we fail to suppress the 5mer. The stop codon needs to be before we find another 5mer. They also wanted us to run through both the RFP and Tet Resistance genes to make sure that they did not contain any of the anticodon sequences from our 5 frameshift suppressor tRNAs so as to throw the translation out of frame. They further wanted us to confirm that if we do have successful suppression, there are no stop codons in the middle of the RFP and Tet Resistance genes. PEARL did not find any matches for the suppressor tRNAs within genes. We found 3 matches for stop codons UAA. Two of these occured at the end of the Tet and one at the end of RFP, where they should normally be to end translation into a protein.

Following this, Olivia Ho-Shing and I tried to determine how we can add BioBrick ends to our single stranded DNA sequences that code for the suppressor tRNAs. Our ultimate goal is to find complements for these single strands so they can be put into plasmids. We found the standard prefixes and suffixes for sequences that do not contain ATG (becuase this sequence is a functional RNA and will not be translated) and we placed these before and after the altered 92bp sequence that Dr. Anderson provided us with. The prefixes and suffixes are necessary because they are the extra nucleotides that will be mimicking a "restriction enzyme cutting site". The single strand appeared as below after the Bio Brick prefixes and suffixes were added:

22ntPREFIX--32nt--VARIABLE ANTICODON LOOP--51nt--21ntSUFFIX

We used the suppressor codon CUAGU as our example and placed its appropriate anticodon loop in the sequence. We put this into the lancelator that would give us the other strand as well as the oligos we would need for ideal construction of this double stranded DNA fragment to put into the plasmid. We recieved a total of 4 oligos, 2 of which were variable and 2 of which were constant for all 6 different suppressor tRNAs. Using this double strand, we simulated a restriction enzyme digestion by EcoRI and PstI. We took off the G at both ends of the top strand and then removed the first five nucleotides on each end of the bottom strand. This made the top strand longer than the bottom one. We then proceeded to take the two variable strands and changed the anticodon loop nt for each suppressor tRNA we used.

Around lunchtime, we encountered a problem with the BioBrick scar in the "ATG-5mer subpiece" of the test project. It turns out that after annealing the sticky ends of the Xba1 and Spe1 sites, the scar created had a stop codon TAG (UAG) that was in frame with the suppressed codon. When the Xba1 site on insert anneals to the Spe1 site on the plasmid (the green portions), we get a scar that reads TACTAG.
[[Image:screen-capture.png]]

For the afternoon, Olivia Ho-Shing and I finished assembly of the oligos of the suppressor tRNA codes. We assembled the document below:

http://gcat.davidson.edu/GcatWiki/images/5/5e/TRNAoligostoOrder.doc

We were assigned tasks of creating "cartoons" to illustrate the phenomenon that had occurred. No one could successfully do this however, because we could not understand how the piece had formed. The parts registry website provided wrong information on the restriction site of Spe1 which slowed our efficiency. This project turned into an effort to try to find two other different restriction enzymes that have complementary sticky ends other than Xba1 and Spe1. We want to get rid oc the scar that contains TACTAG.

We ended up deciding that a hybrid of two ideas was needed: restriction enzymes would need to be used to make several reporter gene sequences with different beginnings dependending on the suppressor tRNA sequence needed.

==Friday, June 5, 2009==

Our goal for this morning was to discuss the hybrid PCR approach to assemble the part that comes before the gene we wanted to express. We decided that we would have a BioBrick prefix and a 5mer followed by a sequence of nucleotides whose identity was up to us. Using PCR we would get the second double strand if we have the following:

BioBrick Prefix--ATG--5mer--gene until we get to restriction site

The PCR primer would attach to the ATG and the 5mer and would replicate the complementary base pairs to make a double stranded DNA sequence. As several of us were confused about PCR methods, we looked at the online link provided by Alyndria Thompson's link to the PCR interactive website which was very helpful. We also did some research to discover that the restriction enzyme BseRI (which is contained in the YFP that we wanted to alter) is not compatible with any other restriction enzyme. This is good because we did not want to plasmid containing this gene to close up with Xba1 once we digested it.

The team experienced some confusion with the hybrid idea and spoke to Clif Davis of Missouri Western University on the phone. He told us that there will be one part that is a fragment of the beginning of some nucleotides as a restriction cleaving site, ATG, the logical clause, and the start of the YFP gene that will be placed into a plasmid containing a space. To the left of the space will be the BioBrick Prefix and the right of the space will contain the rest of the genes in the YFP. Therefore, we decided not to alter any BioBrick ends and just look at restriction sites at the beginning of reporter proteins. The small fragment that will be placed into the plasmid will be made by PCR. The PCR will attach a primer onto the ATG and 5mer and replicate.

While we were discussing this, we expanded upon the idea that we can do whatever we want with the "suffix" portion between the 5mer and the beginning of YFP. We decided it would be best to remove ATG and add the first 5 amino acids and the restriction enzyme site. Along with this, if we have the 5mer coding for a certain amino acid (ie methionine) we could replace another codon that codes for methionine WITH that 5mer so long as this codon is contained within the first 15 nt. This way, we would not be altering the protein.

Olivia Ho-Shing came up with an idea of cascading PCR effect for longer logical clauses. Post translational modification of the protein was another one of her ideas. This applies because although we may code for the protein in a given way, the protein may modify or fix itself up to perform a slightly different function in its tertiary form.

Dr. Campbell suggested that we can look up restriction enzymes 400-500 base pairs into the reporter protein we would like to use; however, we have more flexibility in our procedure if we choose more "hardy" cutters. So instead, we could cut with EcoRI instead of with Xba1.

During the afternoon, we set about three tasks:
1) Which primers to use
2) Which promoters to use
3) Which restriction enzymes and how they will determine use of reporter proteins

Olivia Ho-Shing and I began looking up which restriction enzymes were strong so we could base our reporters on that. We began with Tet and RFP since those were the two reporters we had started out with choosing. I found a website that provided enzyme cleavage efficiency based upon the proximity of the site to the end of the PCR fragment that contains it. We discovered the following based upon the attached documents:

http://gcat.davidson.edu/GcatWiki/images/8/8f/Restriction_Enzyme_Cleavage_Efficiency.doc
http://gcat.davidson.edu/GcatWiki/images/9/9a/07Jun09_RE_Cutting_Efficiency_oh.doc

We decided that these two reporters contained efficient enzymes in a good quantity to choose from. On Monday, we will look through these enzymes once more and finalize which enzymes we would like to use with RFP and Tet.

==Sunday, June 7, 2009==

We did preparations for the mini prep this evening. The following notes are courtesy of Olivia Ho-Shing.

1. Sterically transfer ~2mL LB+AMP (AMP are ampicillin resistant plasmids) *Never put fluid back into bottle once it has entered pipette*
2. Use forceps to grab toothpick, scrap some frozen cells, rub in tube with top

'''Tubes'''

S03511 (2)

K091111

B0030

I715039

K091112

I715039

J31007 (2)

B0034

E1010

S03710

==Monday, June 8, 2009==

We reviewed the PCR process this morning as well as the restriction enzyme cleavage process.

Then, Leland Taylor, Olivia Ho-Shing and I all worked on converting the tRNA oligos we made to the template that Missouri Western used. The oligos we had previously made were different from the ones that Dr. Anderson had originally emailed us because further communication between Dr. Anderson and Missouri Western resulted in a better template.

We tried several times to minimize the number of oligos while keeping their individual lengths under 80bp. We managed to do this in the end by creating 7 oligos, 2 of which are variable between the suppressor tRNAs.
We reviewed the following document and finalized the oligos that we needed to order for the frameshift suppressor tRNAs. We decided to keep both the 10bp and 9bp anticodons of CCAUC.

I also looked up the amino acids that corresponded to the FS (frameshift suppressor) tRNAs for future use:

1. CUAGU = Leu
2. CCCUC = Pro
3. CGGUC = Arg
4. CCACU = Pro
5. CCAUC* = Pro
*9 and 10 bp anticodons

Also, we have decided to use NcoI as the restriction enzyme for RFP. This cleavage site occurs 419 bp into the gene. We have decided to use BamHI as the enzyme for Tetracycline Resistance and the cleavage site occurs 290bp into the gene. Both of these enzymes are very powerful and hardy.

We also looked over Missouri Western's tRNA oligos and primer oligos which we approved.
Our oligos were also approved by Missouri Western and Dr. Campbell ordered them while we supervised.

In the afternoon we did the miniprep. The mini prep directions were as appear in the link below.

http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_MiniPrep.html

We then worked with the Nano Prep to dilute our solution of DNA to an absorption of 1.5 and then we made labels for the tubes. The chart below shows relevant information we obtained from the NanoDrop for each of the reporter plasmids.

[[Image: Screen-capture-6.png]]

==Tuesday, June 9, 2009==

'''Preparations for restriction enzymes digestion of plasmids'''

Purpose: To determine the size of the plasmid inserts and ensure that plasmids are what we extracted from the cells during the mini-prep.

Things we need:
DNA ~3uL
Buffer 10x (Want to dilute to 1x) so add 2uL
Enzyme 1 ~1uL
Enzme 2 ~1uL
Water ~13uL
Final Volunter about 20 uL.

Need to choose the right buffer for the restriction enzymes. All buffers come in 10x (10 times more concentrated than what you want it to be when you use it). [http://gcat.davidson.edu/GcatWiki/index.php/Davidson_Missouri_W/Double_Digest_Guide Buffer Website]

Total volume of enzymes has to be 10% or less than final volume (most volume 2uL). Barely submerge the pipet tip into the solution when pipeting the enzymes.
Star (*) activity: Restriction enzyme stops cutting specifically and starts cutting everywhere.

DNA can't be seen by itself so we add EtBr (the bigger the insert, the more will bind) so bigger strands appear brighter. samples.

The order of entry doesn't matter, but make sure to enter the enzymes last.

Will have lots of labeled tubes and will put 3uL of appropriate plasmids into the tubes.
In the meantime, we'll make a "Master Mix" of all the other ingredients in a bigger tube. Always make enough volume for one extra tube.

Enzymes usually have an optimal temperature of 37C. We will let this reaction go for an hour and then we will run it on a gel.

'''Gel Electrophoresis Protocol'''

Website online for ideal gel concentration for resolving different sizes of molecules [http://gcat.davidson.edu/iGEM08/gelwebsite/gelwebsite.html Gel Concentration Website].
See protocol on the Davidson/Missouri Western Wookie for running molecules in the gel and how to digest with restriction enzymes.

'''What's Next?'''

1. 5' end additions
a. Using PCR
b. Cut at the restriction sites EcoR1 and either Bam H1 or Nco1
c. Cut plasmid and reporter with same restriction enzymes as above
d. Run a gel and purify "keeper" DNA (Ligate transform, screen, sequence)
e. Then, we take the "keeper" DNA and combine it with the insert from part

We can have steps d and e ready in advance.

2. tRNAs
a. Assemble the oligos into genes (Requires lots of calculations beforehand, but the actual steps )
b. Plasmids cut up with EcoR1 and Pst1 '''WE CAN USE THE "WASTE" PLASMID FROM THE RESTRICTION ENZYME DIGEST'''
c. Gel purify the plasmids (Ligate transform, screen, sequence)

The following tables include Master Mix measurements, agarose gel concentrations and buffers.
http://gcat.davidson.edu/GcatWiki/images/0/08/Shamitapic3.png
http://gcat.davidson.edu/GcatWiki/images/d/d1/Shamitapic4.png
http://gcat.davidson.edu/GcatWiki/images/6/6f/Shamitapic5.png

We made the Master Mix with our DNA and put it in the incubator at 10:40 AM.

'''Preparing the Gels'''

Now, we will be making 2 gels, 1 at 0.8% and 2.5% (weight to volume percent). The gel volume is 60mL.

'''Definition of 1% solution:
1 gram of agarose/ 100mL buffer'''

So for a 0.8% solution: 0.8 grams agarose/100mL buffer AND we need 60mL (fixed volume for casts run) so multiply that by 0.6.
Final concentration:

.48 grams agarose/60mL buffer

1 uL of EtBr stock for 60mL of gel.
EtBr is in the buffer so it is constantly replenishing itself even though it is moving toward one end in order to make bands on the other end visible.

After Dr. Campbell's demonstration, we each helped prepare a 0.8% buffer solution using the agarose powder, buffer and EtBr. We mixed together the first two substances in a 200mL Erlenmeyer flask and heated it for 120 seconds in the microwave. We then added EtBr and poured the gel solution into the gel cast.

'''Agar Plate'''

After lunch, we grew some more cells for each of the 11 parts in an agar plate labeled "LBAMP-AMC-9 June" so that so would have extra cells that could be stored for up to 2 weeks.
We made small circles on the agar plate and numbered them 1-11. The numbers corresponded to the following parts. We put the plate in the incubator overnight to let the cells grow.

1. B0030 AMC
2. K091111
3. S03710
4. I715039 OEH
5. S03511 ORH
6. I715039 AST
7. B0030 SDP
8. K091112
9. J31007 LJH
10. J31007 REC
11. S03511

'''Unloading the Gels'''

The following shows which parts we entered in which gels:

[[Image: screen-capture-2.png]]

We unloaded the 2.5% agarose first and after observing the bands under a UV light, put the gel into the BioRad machine to observe the bands. The image below is of a Molecular Weight Marker that shows the number of bp in control fragments of DNA according to the distance they travel down the gel.

[[Image: screen-capture-1.png]]

We obtained the following printout for the first gel that was 2.5% Agarose.
[[Image: 2009-06-09_14hr_44min.jpg]]

The bands in the first well at the bottom are those of the molecular weight marker. We skipped the lane above (to the right) of the MWM and entered the rest of the inserts as described in the above table. A light band is visible for each of the 4 other wells. In Lanes 3 and 4 we concluded we are supposed to have approximately 45 bp because the insert is 15bp + 30 bp on either side of the insert (Xba1 and Spe1 restrictions sites + sticky ends + extra bases). We used the same logic for Lanes 5 and 6 which are supposed to contain approximately 86 base pairs.

One discrepancy in this gel, however, is that Lanes 5 and 6, which according to their labels respectively contain the LacIQ promoter and the Lac IQ1 promoter. According to the information online at the parts registry website, pLacIQ1 should be the same length as pLacIQ. The gels do not show this, but instead show that the sample in Lane 5 is smaller than that in Lane 6 because Lane 5 traveled further. Leland Taylor and Romina Clemente sent out an email to Pallavi Penumetcha to ask about this because she had written a paper about this.

We then unloaded the 0.8% agarose gel and then placed this in the BioRad machine under the same protocol as above. The following image is the 0.8% Agarose.

[[Image:2009-06-09_15hr_21min.jpg]]

We observed that the bands in Lane 1 (which is now at the top) are examples of high intensity bands. The plasmids contained in the original well are twice the intensity of the insert.

In Lane 5, however, we observed several different bands. This could be due to the extra DNA that was sitting on the top of the well. We decided to re-expose the gel to get better observations on Lanes 3 and 4 because the bands were not as intense. We anticipated seeing a band at about 250 bp because this was the size of the insert (plus extra bp). After re-exposing the gel, we did not find any difference and still did not see any bands in these lanes. We have decided to redo the gel on the part S03511 and so we prepared the master mix with that DNA and incubated it so that we could run the gel tomorrow.

We also cut out the "empty" plasmids from the gel so that we could use this as vectors for the coding sequence of the suppressor tRNAs.

Tomorrow we will be planning out specific tasks for people once the oligos arrive.

==Wednesday, June 10, 2009==

We began this morning by making the agarose gel with the same protocol as yesterday. This time, since we are just re-doing both S03511 inserts, we will have a 1.8% agarose gel.

'''OUTLINE: WHERE DO WE STAND?'''

Yesterday
a. Isolated empty plasmids
b. Verified most inserts
c. Waiting for:
>tRNA oligos
>Primers
a. Will amplify the reporter gene in the plasmid itself

Next?
a. When Primers Arrive
> Amplify the reporter genes with primers (PCR takes ~3 hours)
> Calculate how much reporter plasmid we will need (Start with 50x more than we actually will need)
> Cut amplified sequence (EcoR1 and BamH1/Nco1) then purify what we need
> Cut reporter plasmid (EcoR1 and BamH1/Nco1) then purify what we need
> Ligate amplified sequence and reporter plasmid
b. When tRNA oligos arrive
> Calculate how much of each oligo we will need (takes a while...)
> "Boil and cool" tRNA oligos together
> Ligate fragments (dsDNA) into plasmids we isolated yesterday (~5 minutes)
> Transform the plasmids (~20 minutes)

* LOOK THROUGH LIGATION PROTOCOL (How to assemble genes)
* LOOK THROUGH TRANSFORMATION PROTOCOL

''What we need to get done today'':
1. Prepare the control for the Tet Resistance by BioBricking the part pLac+RBS (S03511) with the TetA (J31007).
2. Check the part number for RFP and also for pLac+RBS+RFP
3. Read through ligation protocol
4. See how many nanograms of plasmid we will need for the tRNAs and for the inserts and multiply this number by 50.
> Check if we have enough plasmids from our mini-prep
5. Purify the plasmids from the gel

After we made this outline, Leland, Olivia and I analyzed the gel we ran this morning and did not see a more than one band in the 2 lanes that contained DNA from S03511 (image below). Since we did not see multiple bands in yesterday's gel either, we conclude that there is something wrong with the DNA rather than with the restriction enzymes that we used to cut it. We concluded that there is probably not an insert in this plasmid both the part and the plasmid are useless.

[[Image: 2009-06-10 10hr 30min.jpg]]

Now, we do not have an immediately accessible promoter. We have decided to order the pBAD promoter from Missouri Western and we will attach the RBS+Tet to this plasmid. Our insert for this will be the larger gene because it is easier to manipulate in the gel than the small gene.

Alyndria Thompson and Romina Clemente are figuring out the size of the plasmids including the reporter genes.

After lunch we performed several steps to isolate and purify the gel to get the empty plasmids will be the vectors for the tRNA DNA. Then, we used the NanoDrop to assess the purity of our DNA. We found that the plasmids were more pure than we had expected. The following values were obtained:

Abs 1.850
A-260 10 mm path 0.172
A-280 10 mm path 0.075
260/280 2.28
260/230 0.09

We then followed the protocol for preparing the agar plates. We put the plate with the agar solution in the Autoclave.

While the solution was in the autoclave, we discussed that we don't really need a promoter in front of RBS+Tet for the gene to be expressed because Tet expression is so strong that the gene would translate without a promoter. However, it would be a good idea to have the promoter because if it was possible to reinforce the translation of the reporter protein, we wanted to do that.

We found that there were some tubes in the lab fridge that contained the pBAD and pLac promoters with RBS. We decided to use these as the promoter and RBS for our experiments. The pLac promoter will be used for RFP and the pBad promoter used with Tet Resistance. We decided to use the pBad with Tet because it is weaker in comparison to pLac. As expression of the Tet gene can be toxic to a cell, we do not want to overexpress the gene with a stronger promoter.

Once the agar solution was sterilized in the Autoclave, we removed it, cooled it, and pipetted the antibiotic resistance into it. We then poured the solution into the plates, removed any bubbles, and let the gel form.

Alyndria Thompson and Romina Clemente concluded that we had more than enough recieving vectors for both the RFP and Tetracycline genes. Since we need 50 nanograms, it is wise to have at least 50 times that much that we can work with, so we need at least 2,500 ng.

RFP 1: 77.6 x 4 = 310.4 ng/uL
x 20 uL = 3,272 ng
RFP 2: 59.5 x 4 = 238 ng/uL
x 20 uL = 4,760 ng
Tet: 40.9 x 4 = 163.6 ng/uL
x 20 uL = 3,272 ng

According to these calculations we have more than 50 times what we need for both instances of RFP and Tet.

Tomorrow, we can prepare the Master Mix for the digestions of both reporter plamids and then add the restriction enzymes once they arrive. Then, we can follow through with the rest of the plans we had made above in the section titles "Next?".

==Thursday, June 11, 2009==

We began this morning by calculating how many uL we would need for each of the mixes for digesting our reporter plasmids. We decided to double the final volume of each mix to 40uL instead of 20uL. Since we knew the concentration of each plasmid and also knew we needed 2500 ng, we cross multiplied to find the uL of DNA we would need for each.

Concentration Calculations:
310.4 ng/1 uL = 2500ng/x uL
x = 8.05 uL

Here is a chart indicating how much of each we used for the 2 mixes.

[[Image: DoubledigestRFPTetR.png]]

On the Promega website, we found a list of compatible buffers for double digestions. Buffers E and H are compatible for the Tet plasmid and RFP plasmid digestions respectively. We fortunately have both buffers in stock.

[[Image:BamEcoR1.png]] [[Image:NcoEcoR1.png]]

We waited for our waited for the restriction enzymes to arrive and in the meantime, we transformed the plasmids with pLac+RBS, pBad, and pBad+RBS into ''E.coli'' cells. We want the ''E.coli'' cells to replicate the plasmids with these genes because we have a limited amount of genes. [http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_Transformation.html Zippy Plasmid Transformation]

Once our oligos and restriction enzymes arrived, we got to work right away!

'''PCR (Polymerase Chain Reaction)'''

We calculated how much plasmid we will need for the PCR. We decided to dilute the concentration of our plasmids a hundred fold so that we could get pipetable amounts.

''Change in Concentrations''
310.4 ng/uL x 1/100 (dilution factor) = 3.104 ng/uL (RFP)
163.6 ng/uL x 1/100 (dilution factor) = 0.164 ng/uL (Tet)

''Example shown with RFP''
3.104 ng/1 uL = 1ng/ x uL, x = .32
For Tet, x = .61

We also calculated how much of each primer we would need with the following equation:

C1V1 = C2V2
(100 uM) (x) = (1 uM)( 100 uL)
x = 1 uL

[[Image: screen-capture-8.png]]

Each of the 5 of us made had 2 tubes (one for each reporter protein) because there were 5 different forward primers.To each tube, we added the template DNA (either RFP or Tet), water, the respective reverse primer (either RFP or Tet), and the forward primer depending on which 5mer we had chosen. My 5mer was CCAUC. Finally, we added the Master Mix last because this contained the enzyme (DNA polymerase) that would amplify our desired sequence.

[[Image: Pcrtubes.png]]

We followed the temperature cycles so that PCR could occur. The annealing temperature needed to be 5C less than the smallest melting temperature of the primers. Since the reverse primer of RFP had a melting temp of 66C, we set the temperature of the 3rd cycle on 61C.

[[Image: Temperaturecycle.png]]

'''tRNA Oligo Assembly'''

We needed to calculate the concentration of each of the oligo before carrying out the assembly. The stock concentration of each of the oligo was 100 uM. Since we wanted to have 5uM of the oligo in the test tube, and we know that final volume of the test tube is 20 uL, we just cross multiplied to find the amount we needed to add to the tubes to get this concentration.

(100uM)x = (5uM)(20uL)
x = 1 uL

We added 1 uL of each oligo to the tubes.

We had six total tRNAs we needed to assemble so each of us took one FML (one person took two) and to each tube we added 1uL of each oligo for a total of 7uL, 2uL of buffer, and 11uL of water for a total volume of 20 uL. Five of the oligos were constant and 2 varied for each of the different tRNAs. So everyone pipetted the same 5 oligos and everyone also had 2 unique oligos to add. When all of the oligos were added and the tubes ready, we boiled the tubes at 50C for 5 minutes. We then allowed the tubes to remain in the water to cool very slowly overnight so that the annealing process could take place.

The last thing we did at the end of the day was finish off our plasmid digestion that we had begun in the morning. We added the buffer, DNA, water, and restriction enzymes and placed this in the incubator for overnight digestion.

'''To Do''':

1. Isolate our desired dsDNA from the PCR.
2. Verify our tRNA inserts and eventually transform these into plasmids.
3. Check our agar plates to see colonies that have replicated our pLac+RBS, pBAD+RBS, and pBAD genes.

==Friday, June 12, 2009==

This morning, we began by outlining the main items on today's agenda.

1. tRNAs: Ligation, Transformation
2. Digestion reporters (run all of it and gel purify)
3. Transformed cells
4. PCR: Gel run an aliquot to verify (5uL)

Shamita Punjabi Notebook

2009-06-12T02:01:38Z

ShPunjabi:

[[iGEM Notebook]]

Shamita Punjabi Notebook

2009-06-12T01:54:00Z

ShPunjabi:

[[iGEM Notebook June/July 2009]]

IGEM Notebook

2009-06-12T01:50:19Z

ShPunjabi: /* Thursday, June 11, 2009 */

==Wednesday, June 3, 2009==

Romina Clemente and I are trying to find suitable reporter proteins to use. Yesterday, Leland Taylor and Alyndria Thompson were working on ways to insert the gene sequences into the plasmid. Upon seeing how they wanted to manipulated the reporter gene to include the logical clauses, we came up with a few criteria for the reporter genes we would use. The following criteria for genes are listed in order of the broadest aspect to look at to the narrowest aspect:
a) Doesn't contain restriction sites for the 4 restriction enzymes (EcoR1, Xbal, Spel, Pst1) used to cleave the Biobrick part out of the plasmid.
b) Contains 6 cutter restriction sites.
c) These restriction enzymes aren't blunt (cleave straight down at one spot).
d) These restriction sites are close to thge 5' (beginning) end of the sequence.
e) These enzymes are easiest to work with and cheapest.
We are finding the part numbers of the reporter genes we want to use (antibiotic resistance, fluorescence, LacZ) through our own GCAT because we know these ones work. We are then locating these parts on the parts registry [1] website. We copied and pasted the gene sequences we obtained from the registry onto the ApE software [2]. From here, we were able to generate a genetic map of each gene that outlined each restriction site that fit our criteria. We put each genetic map, alongside the part number used, into a Word document.

http://gcat.davidson.edu/GcatWiki/images/0/0e/Restriction_Site_Mapping_on_Reporter_Genes.doc

Later on in the day, we decided that the best way to test for suppression would involve placing the 5mers at the beginning of the reporter gene instead of inserting them into the reporter gene. To do this, we decided that the gene along with its start codon need to be expressed after the 5mer. We will insert a BioBrick plasmid with a 5mer and start codon that will be incorporated before the gene:
RBS-6-8nt-ATG-5mer--ATG-gene

Leland Taylor assembled the oligos that we will need for the BioBrick pieces.

In order to test whether the reporter protein would be expressed regardless of the 5mer being suppressed or not, we would need to remove the suppressor tRNAs from the cell.

Meanwhile, I helped figure out the coding sequence that we will need for the suppressor tRNAs that will bind to the 5mers. We decided that we would be using the following suppressor tRNAs on the Davidson side: CUAGU, CCCUC, CGGUC, CCAUC, and CCACU. We searched through several papers, with little result and finally emailed Dr. Christopher Anderson of UC Berkeley to request the DNA sequence. He emailed us back with a generic DNA code that we could use to create the tRNA; however, for each suppressor tRNA we would use, we would need to change base pairs in the anticodon loop.

The length of the tRNA is 92 nt. The anticodon loop is comprised of 9 nt. Once we have BioBricked the DNA sequence with 4 restriction sites and supplemental nucleotides, the length of the entire gene is 144 nucleotides. This is made up of the 92 nt +22 nt on each side of the sequence (44) + 4 nt on each side of the restriction ends (8).

At the end of the day, we decided to use tetracycline resistance and RFP as our reporter proteins for this tester experiment.

==Thursday, June 4, 2009==

This morning, I began the day by learning how to use the PEARL prgram. Olivia Ho-Shing sat with me and walked me through the program script. And then, Leland Taylor and Shashank Suresh had a problem they wanted us to solve. Leland explained that we need to make sure there is a stop codon in the gene if we fail to suppress the 5mer. The stop codon needs to be before we find another 5mer. They also wanted us to run through both the RFP and Tet Resistance genes to make sure that they did not contain any of the anticodon sequences from our 5 frameshift suppressor tRNAs so as to throw the translation out of frame. They further wanted us to confirm that if we do have successful suppression, there are no stop codons in the middle of the RFP and Tet Resistance genes. PEARL did not find any matches for the suppressor tRNAs within genes. We found 3 matches for stop codons UAA. Two of these occured at the end of the Tet and one at the end of RFP, where they should normally be to end translation into a protein.

Following this, Olivia Ho-Shing and I tried to determine how we can add BioBrick ends to our single stranded DNA sequences that code for the suppressor tRNAs. Our ultimate goal is to find complements for these single strands so they can be put into plasmids. We found the standard prefixes and suffixes for sequences that do not contain ATG (becuase this sequence is a functional RNA and will not be translated) and we placed these before and after the altered 92bp sequence that Dr. Anderson provided us with. The prefixes and suffixes are necessary because they are the extra nucleotides that will be mimicking a "restriction enzyme cutting site". The single strand appeared as below after the Bio Brick prefixes and suffixes were added:

22ntPREFIX--32nt--VARIABLE ANTICODON LOOP--51nt--21ntSUFFIX

We used the suppressor codon CUAGU as our example and placed its appropriate anticodon loop in the sequence. We put this into the lancelator that would give us the other strand as well as the oligos we would need for ideal construction of this double stranded DNA fragment to put into the plasmid. We recieved a total of 4 oligos, 2 of which were variable and 2 of which were constant for all 6 different suppressor tRNAs. Using this double strand, we simulated a restriction enzyme digestion by EcoRI and PstI. We took off the G at both ends of the top strand and then removed the first five nucleotides on each end of the bottom strand. This made the top strand longer than the bottom one. We then proceeded to take the two variable strands and changed the anticodon loop nt for each suppressor tRNA we used.

Around lunchtime, we encountered a problem with the BioBrick scar in the "ATG-5mer subpiece" of the test project. It turns out that after annealing the sticky ends of the Xba1 and Spe1 sites, the scar created had a stop codon TAG (UAG) that was in frame with the suppressed codon. When the Xba1 site on insert anneals to the Spe1 site on the plasmid (the green portions), we get a scar that reads TACTAG.
[[Image:screen-capture.png]]

For the afternoon, Olivia Ho-Shing and I finished assembly of the oligos of the suppressor tRNA codes. We assembled the document below:

http://gcat.davidson.edu/GcatWiki/images/5/5e/TRNAoligostoOrder.doc

We were assigned tasks of creating "cartoons" to illustrate the phenomenon that had occurred. No one could successfully do this however, because we could not understand how the piece had formed. The parts registry website provided wrong information on the restriction site of Spe1 which slowed our efficiency. This project turned into an effort to try to find two other different restriction enzymes that have complementary sticky ends other than Xba1 and Spe1. We want to get rid oc the scar that contains TACTAG.

We ended up deciding that a hybrid of two ideas was needed: restriction enzymes would need to be used to make several reporter gene sequences with different beginnings dependending on the suppressor tRNA sequence needed.

==Friday, June 5, 2009==

Our goal for this morning was to discuss the hybrid PCR approach to assemble the part that comes before the gene we wanted to express. We decided that we would have a BioBrick prefix and a 5mer followed by a sequence of nucleotides whose identity was up to us. Using PCR we would get the second double strand if we have the following:

BioBrick Prefix--ATG--5mer--gene until we get to restriction site

The PCR primer would attach to the ATG and the 5mer and would replicate the complementary base pairs to make a double stranded DNA sequence. As several of us were confused about PCR methods, we looked at the online link provided by Alyndria Thompson's link to the PCR interactive website which was very helpful. We also did some research to discover that the restriction enzyme BseRI (which is contained in the YFP that we wanted to alter) is not compatible with any other restriction enzyme. This is good because we did not want to plasmid containing this gene to close up with Xba1 once we digested it.

The team experienced some confusion with the hybrid idea and spoke to Clif Davis of Missouri Western University on the phone. He told us that there will be one part that is a fragment of the beginning of some nucleotides as a restriction cleaving site, ATG, the logical clause, and the start of the YFP gene that will be placed into a plasmid containing a space. To the left of the space will be the BioBrick Prefix and the right of the space will contain the rest of the genes in the YFP. Therefore, we decided not to alter any BioBrick ends and just look at restriction sites at the beginning of reporter proteins. The small fragment that will be placed into the plasmid will be made by PCR. The PCR will attach a primer onto the ATG and 5mer and replicate.

While we were discussing this, we expanded upon the idea that we can do whatever we want with the "suffix" portion between the 5mer and the beginning of YFP. We decided it would be best to remove ATG and add the first 5 amino acids and the restriction enzyme site. Along with this, if we have the 5mer coding for a certain amino acid (ie methionine) we could replace another codon that codes for methionine WITH that 5mer so long as this codon is contained within the first 15 nt. This way, we would not be altering the protein.

Olivia Ho-Shing came up with an idea of cascading PCR effect for longer logical clauses. Post translational modification of the protein was another one of her ideas. This applies because although we may code for the protein in a given way, the protein may modify or fix itself up to perform a slightly different function in its tertiary form.

Dr. Campbell suggested that we can look up restriction enzymes 400-500 base pairs into the reporter protein we would like to use; however, we have more flexibility in our procedure if we choose more "hardy" cutters. So instead, we could cut with EcoRI instead of with Xba1.

During the afternoon, we set about three tasks:
1) Which primers to use
2) Which promoters to use
3) Which restriction enzymes and how they will determine use of reporter proteins

Olivia Ho-Shing and I began looking up which restriction enzymes were strong so we could base our reporters on that. We began with Tet and RFP since those were the two reporters we had started out with choosing. I found a website that provided enzyme cleavage efficiency based upon the proximity of the site to the end of the PCR fragment that contains it. We discovered the following based upon the attached documents:

http://gcat.davidson.edu/GcatWiki/images/8/8f/Restriction_Enzyme_Cleavage_Efficiency.doc
http://gcat.davidson.edu/GcatWiki/images/9/9a/07Jun09_RE_Cutting_Efficiency_oh.doc

We decided that these two reporters contained efficient enzymes in a good quantity to choose from. On Monday, we will look through these enzymes once more and finalize which enzymes we would like to use with RFP and Tet.

==Sunday, June 7, 2009==

We did preparations for the mini prep this evening. The following notes are courtesy of Olivia Ho-Shing.

1. Sterically transfer ~2mL LB+AMP (AMP are ampicillin resistant plasmids) *Never put fluid back into bottle once it has entered pipette*
2. Use forceps to grab toothpick, scrap some frozen cells, rub in tube with top

'''Tubes'''

S03511 (2)

K091111

B0030

I715039

K091112

I715039

J31007 (2)

B0034

E1010

S03710

==Monday, June 8, 2009==

We reviewed the PCR process this morning as well as the restriction enzyme cleavage process.

Then, Leland Taylor, Olivia Ho-Shing and I all worked on converting the tRNA oligos we made to the template that Missouri Western used. The oligos we had previously made were different from the ones that Dr. Anderson had originally emailed us because further communication between Dr. Anderson and Missouri Western resulted in a better template.

We tried several times to minimize the number of oligos while keeping their individual lengths under 80bp. We managed to do this in the end by creating 7 oligos, 2 of which are variable between the suppressor tRNAs.
We reviewed the following document and finalized the oligos that we needed to order for the frameshift suppressor tRNAs. We decided to keep both the 10bp and 9bp anticodons of CCAUC.

I also looked up the amino acids that corresponded to the FS (frameshift suppressor) tRNAs for future use:

1. CUAGU = Leu
2. CCCUC = Pro
3. CGGUC = Arg
4. CCACU = Pro
5. CCAUC* = Pro
*9 and 10 bp anticodons

Also, we have decided to use NcoI as the restriction enzyme for RFP. This cleavage site occurs 419 bp into the gene. We have decided to use BamHI as the enzyme for Tetracycline Resistance and the cleavage site occurs 290bp into the gene. Both of these enzymes are very powerful and hardy.

We also looked over Missouri Western's tRNA oligos and primer oligos which we approved.
Our oligos were also approved by Missouri Western and Dr. Campbell ordered them while we supervised.

In the afternoon we did the miniprep. The mini prep directions were as appear in the link below.

http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_MiniPrep.html

We then worked with the Nano Prep to dilute our solution of DNA to an absorption of 1.5 and then we made labels for the tubes. The chart below shows relevant information we obtained from the NanoDrop for each of the reporter plasmids.

[[Image: Screen-capture-6.png]]

==Tuesday, June 9, 2009==

'''Preparations for restriction enzymes digestion of plasmids'''

Purpose: To determine the size of the plasmid inserts and ensure that plasmids are what we extracted from the cells during the mini-prep.

Things we need:
DNA ~3uL
Buffer 10x (Want to dilute to 1x) so add 2uL
Enzyme 1 ~1uL
Enzme 2 ~1uL
Water ~13uL
Final Volunter about 20 uL.

Need to choose the right buffer for the restriction enzymes. All buffers come in 10x (10 times more concentrated than what you want it to be when you use it). [http://gcat.davidson.edu/GcatWiki/index.php/Davidson_Missouri_W/Double_Digest_Guide Buffer Website]

Total volume of enzymes has to be 10% or less than final volume (most volume 2uL). Barely submerge the pipet tip into the solution when pipeting the enzymes.
Star (*) activity: Restriction enzyme stops cutting specifically and starts cutting everywhere.

DNA can't be seen by itself so we add EtBr (the bigger the insert, the more will bind) so bigger strands appear brighter. samples.

The order of entry doesn't matter, but make sure to enter the enzymes last.

Will have lots of labeled tubes and will put 3uL of appropriate plasmids into the tubes.
In the meantime, we'll make a "Master Mix" of all the other ingredients in a bigger tube. Always make enough volume for one extra tube.

Enzymes usually have an optimal temperature of 37C. We will let this reaction go for an hour and then we will run it on a gel.

'''Gel Electrophoresis Protocol'''

Website online for ideal gel concentration for resolving different sizes of molecules [http://gcat.davidson.edu/iGEM08/gelwebsite/gelwebsite.html Gel Concentration Website].
See protocol on the Davidson/Missouri Western Wookie for running molecules in the gel and how to digest with restriction enzymes.

'''What's Next?'''

1. 5' end additions
a. Using PCR
b. Cut at the restriction sites EcoR1 and either Bam H1 or Nco1
c. Cut plasmid and reporter with same restriction enzymes as above
d. Run a gel and purify "keeper" DNA (Ligate transform, screen, sequence)
e. Then, we take the "keeper" DNA and combine it with the insert from part

We can have steps d and e ready in advance.

2. tRNAs
a. Assemble the oligos into genes (Requires lots of calculations beforehand, but the actual steps )
b. Plasmids cut up with EcoR1 and Pst1 '''WE CAN USE THE "WASTE" PLASMID FROM THE RESTRICTION ENZYME DIGEST'''
c. Gel purify the plasmids (Ligate transform, screen, sequence)

The following tables include Master Mix measurements, agarose gel concentrations and buffers.
http://gcat.davidson.edu/GcatWiki/images/0/08/Shamitapic3.png
http://gcat.davidson.edu/GcatWiki/images/d/d1/Shamitapic4.png
http://gcat.davidson.edu/GcatWiki/images/6/6f/Shamitapic5.png

We made the Master Mix with our DNA and put it in the incubator at 10:40 AM.

'''Preparing the Gels'''

Now, we will be making 2 gels, 1 at 0.8% and 2.5% (weight to volume percent). The gel volume is 60mL.

'''Definition of 1% solution:
1 gram of agarose/ 100mL buffer'''

So for a 0.8% solution: 0.8 grams agarose/100mL buffer AND we need 60mL (fixed volume for casts run) so multiply that by 0.6.
Final concentration:

.48 grams agarose/60mL buffer

1 uL of EtBr stock for 60mL of gel.
EtBr is in the buffer so it is constantly replenishing itself even though it is moving toward one end in order to make bands on the other end visible.

After Dr. Campbell's demonstration, we each helped prepare a 0.8% buffer solution using the agarose powder, buffer and EtBr. We mixed together the first two substances in a 200mL Erlenmeyer flask and heated it for 120 seconds in the microwave. We then added EtBr and poured the gel solution into the gel cast.

'''Agar Plate'''

After lunch, we grew some more cells for each of the 11 parts in an agar plate labeled "LBAMP-AMC-9 June" so that so would have extra cells that could be stored for up to 2 weeks.
We made small circles on the agar plate and numbered them 1-11. The numbers corresponded to the following parts. We put the plate in the incubator overnight to let the cells grow.

1. B0030 AMC
2. K091111
3. S03710
4. I715039 OEH
5. S03511 ORH
6. I715039 AST
7. B0030 SDP
8. K091112
9. J31007 LJH
10. J31007 REC
11. S03511

'''Unloading the Gels'''

The following shows which parts we entered in which gels:

[[Image: screen-capture-2.png]]

We unloaded the 2.5% agarose first and after observing the bands under a UV light, put the gel into the BioRad machine to observe the bands. The image below is of a Molecular Weight Marker that shows the number of bp in control fragments of DNA according to the distance they travel down the gel.

[[Image: screen-capture-1.png]]

We obtained the following printout for the first gel that was 2.5% Agarose.
[[Image: 2009-06-09_14hr_44min.jpg]]

The bands in the first well at the bottom are those of the molecular weight marker. We skipped the lane above (to the right) of the MWM and entered the rest of the inserts as described in the above table. A light band is visible for each of the 4 other wells. In Lanes 3 and 4 we concluded we are supposed to have approximately 45 bp because the insert is 15bp + 30 bp on either side of the insert (Xba1 and Spe1 restrictions sites + sticky ends + extra bases). We used the same logic for Lanes 5 and 6 which are supposed to contain approximately 86 base pairs.

One discrepancy in this gel, however, is that Lanes 5 and 6, which according to their labels respectively contain the LacIQ promoter and the Lac IQ1 promoter. According to the information online at the parts registry website, pLacIQ1 should be the same length as pLacIQ. The gels do not show this, but instead show that the sample in Lane 5 is smaller than that in Lane 6 because Lane 5 traveled further. Leland Taylor and Romina Clemente sent out an email to Pallavi Penumetcha to ask about this because she had written a paper about this.

We then unloaded the 0.8% agarose gel and then placed this in the BioRad machine under the same protocol as above. The following image is the 0.8% Agarose.

[[Image:2009-06-09_15hr_21min.jpg]]

We observed that the bands in Lane 1 (which is now at the top) are examples of high intensity bands. The plasmids contained in the original well are twice the intensity of the insert.

In Lane 5, however, we observed several different bands. This could be due to the extra DNA that was sitting on the top of the well. We decided to re-expose the gel to get better observations on Lanes 3 and 4 because the bands were not as intense. We anticipated seeing a band at about 250 bp because this was the size of the insert (plus extra bp). After re-exposing the gel, we did not find any difference and still did not see any bands in these lanes. We have decided to redo the gel on the part S03511 and so we prepared the master mix with that DNA and incubated it so that we could run the gel tomorrow.

We also cut out the "empty" plasmids from the gel so that we could use this as vectors for the coding sequence of the suppressor tRNAs.

Tomorrow we will be planning out specific tasks for people once the oligos arrive.

==Wednesday, June 10, 2009==

We began this morning by making the agarose gel with the same protocol as yesterday. This time, since we are just re-doing both S03511 inserts, we will have a 1.8% agarose gel.

'''OUTLINE: WHERE DO WE STAND?'''

Yesterday
a. Isolated empty plasmids
b. Verified most inserts
c. Waiting for:
>tRNA oligos
>Primers
a. Will amplify the reporter gene in the plasmid itself

Next?
a. When Primers Arrive
> Amplify the reporter genes with primers (PCR takes ~3 hours)
> Calculate how much reporter plasmid we will need (Start with 50x more than we actually will need)
> Cut amplified sequence (EcoR1 and BamH1/Nco1) then purify what we need
> Cut reporter plasmid (EcoR1 and BamH1/Nco1) then purify what we need
> Ligate amplified sequence and reporter plasmid
b. When tRNA oligos arrive
> Calculate how much of each oligo we will need (takes a while...)
> "Boil and cool" tRNA oligos together
> Ligate fragments (dsDNA) into plasmids we isolated yesterday (~5 minutes)
> Transform the plasmids (~20 minutes)

* LOOK THROUGH LIGATION PROTOCOL (How to assemble genes)
* LOOK THROUGH TRANSFORMATION PROTOCOL

''What we need to get done today'':
1. Prepare the control for the Tet Resistance by BioBricking the part pLac+RBS (S03511) with the TetA (J31007).
2. Check the part number for RFP and also for pLac+RBS+RFP
3. Read through ligation protocol
4. See how many nanograms of plasmid we will need for the tRNAs and for the inserts and multiply this number by 50.
> Check if we have enough plasmids from our mini-prep
5. Purify the plasmids from the gel

After we made this outline, Leland, Olivia and I analyzed the gel we ran this morning and did not see a more than one band in the 2 lanes that contained DNA from S03511 (image below). Since we did not see multiple bands in yesterday's gel either, we conclude that there is something wrong with the DNA rather than with the restriction enzymes that we used to cut it. We concluded that there is probably not an insert in this plasmid both the part and the plasmid are useless.

[[Image: 2009-06-10 10hr 30min.jpg]]

Now, we do not have an immediately accessible promoter. We have decided to order the pBAD promoter from Missouri Western and we will attach the RBS+Tet to this plasmid. Our insert for this will be the larger gene because it is easier to manipulate in the gel than the small gene.

Alyndria Thompson and Romina Clemente are figuring out the size of the plasmids including the reporter genes.

After lunch we performed several steps to isolate and purify the gel to get the empty plasmids will be the vectors for the tRNA DNA. Then, we used the NanoDrop to assess the purity of our DNA. We found that the plasmids were more pure than we had expected. The following values were obtained:

Abs 1.850
A-260 10 mm path 0.172
A-280 10 mm path 0.075
260/280 2.28
260/230 0.09

We then followed the protocol for preparing the agar plates. We put the plate with the agar solution in the Autoclave.

While the solution was in the autoclave, we discussed that we don't really need a promoter in front of RBS+Tet for the gene to be expressed because Tet expression is so strong that the gene would translate without a promoter. However, it would be a good idea to have the promoter because if it was possible to reinforce the translation of the reporter protein, we wanted to do that.

We found that there were some tubes in the lab fridge that contained the pBAD and pLac promoters with RBS. We decided to use these as the promoter and RBS for our experiments. The pLac promoter will be used for RFP and the pBad promoter used with Tet Resistance. We decided to use the pBad with Tet because it is weaker in comparison to pLac. As expression of the Tet gene can be toxic to a cell, we do not want to overexpress the gene with a stronger promoter.

Once the agar solution was sterilized in the Autoclave, we removed it, cooled it, and pipetted the antibiotic resistance into it. We then poured the solution into the plates, removed any bubbles, and let the gel form.

Alyndria Thompson and Romina Clemente concluded that we had more than enough recieving vectors for both the RFP and Tetracycline genes. Since we need 50 nanograms, it is wise to have at least 50 times that much that we can work with, so we need at least 2,500 ng.

RFP 1: 77.6 x 4 = 310.4 ng/uL
x 20 uL = 3,272 ng
RFP 2: 59.5 x 4 = 238 ng/uL
x 20 uL = 4,760 ng
Tet: 40.9 x 4 = 163.6 ng/uL
x 20 uL = 3,272 ng

According to these calculations we have more than 50 times what we need for both instances of RFP and Tet.

Tomorrow, we can prepare the Master Mix for the digestions of both reporter plamids and then add the restriction enzymes once they arrive. Then, we can follow through with the rest of the plans we had made above in the section titles "Next?".

==Thursday, June 11, 2009==

We began this morning by calculating how many uL we would need for each of the mixes for digesting our reporter plasmids. We decided to double the final volume of each mix to 40uL instead of 20uL. Since we knew the concentration of each plasmid and also knew we needed 2500 ng, we cross multiplied to find the uL of DNA we would need for each.

Concentration Calculations:
310.4 ng/1 uL = 2500ng/x uL
x = 8.05 uL

Here is a chart indicating how much of each we used for the 2 mixes.

[[Image: DoubledigestRFPTetR.png]]

On the Promega website, we found a list of compatible buffers for double digestions. Buffers E and H are compatible for the Tet plasmid and RFP plasmid digestions respectively. We fortunately have both buffers in stock.

[[Image:BamEcoR1.png]] [[Image:NcoEcoR1.png]]

We waited for our waited for the restriction enzymes to arrive and in the meantime, we transformed the plasmids with pLac+RBS, pBad, and pBad+RBS into ''E.coli'' cells. We want the ''E.coli'' cells to replicate the plasmids with these genes because we have a limited amount of genes. [http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_Transformation.html Zippy Plasmid Transformation]

Once our oligos and restriction enzymes arrived, we got to work right away!

'''PCR (Polymerase Chain Reaction)'''

We calculated how much plasmid we will need for the PCR. We decided to dilute the concentration of our plasmids a hundred fold so that we could get pipetable amounts.

''Change in Concentrations''
310.4 ng/uL x 1/100 (dilution factor) = 3.104 ng/uL (RFP)
163.6 ng/uL x 1/100 (dilution factor) = 0.164 ng/uL (Tet)

''Example shown with RFP''
3.104 ng/1 uL = 1ng/ x uL, x = .32
For Tet, x = .61

We also calculated how much of each primer we would need with the following equation:

C1V1 = C2V2
(100 uM) (x) = (1 uM)( 100 uL)
x = 1 uL

[[Image: screen-capture-8.png]]

Each of the 5 of us made had 2 tubes (one for each reporter protein) because there were 5 different forward primers.To each tube, we added the template DNA (either RFP or Tet), water, the respective reverse primer (either RFP or Tet), and the forward primer depending on which 5mer we had chosen. My 5mer was CCAUC. Finally, we added the Master Mix last because this contained the enzyme (DNA polymerase) that would amplify our desired sequence.

[[Image: Pcrtubes.png]]

We followed the temperature cycles so that PCR could occur. The annealing temperature needed to be 5C less than the smallest melting temperature of the primers. Since the reverse primer of RFP had a melting temp of 66C, we set the temperature of the 3rd cycle on 61C.

[[Image: Temperaturecycle.png]]

'''tRNA Oligo Assembly'''

We needed to calculate the concentration of each of the oligo before carrying out the assembly. The stock concentration of each of the oligo was 100 uM. Since we wanted to have 5uM of the oligo in the test tube, and we know that final volume of the test tube is 20 uL, we just cross multiplied to find the amount we needed to add to the tubes to get this concentration.

(100uM)x = (5uM)(20uL)
x = 1 uL

We added 1 uL of each oligo to the tubes.

We had six total tRNAs we needed to assemble so each of us took one FML (one person took two) and to each tube we added 1uL of each oligo for a total of 7uL, 2uL of buffer, and 11uL of water for a total volume of 20 uL. Five of the oligos were constant and 2 varied for each of the different tRNAs. So everyone pipetted the same 5 oligos and everyone also had 2 unique oligos to add. When all of the oligos were added and the tubes ready, we boiled the tubes at 50C for 5 minutes. We then allowed the tubes to remain in the water to cool very slowly overnight so that the annealing process could take place.

The last thing we did at the end of the day was finish off our plasmid digestion that we had begun in the morning. We added the buffer, DNA, water, and restriction enzymes and placed this in the incubator for overnight digestion.

'''To Do''':

1. Isolate our desired dsDNA from the PCR.
2. Verify our tRNA inserts and eventually transform these into plasmids.
3. Check our agar plates to see colonies that have replicated our pLac+RBS, pBAD+RBS, and pBAD genes.

IGEM Notebook

2009-06-12T01:39:53Z

ShPunjabi: /* Thursday, June 11, 2009 */

==Wednesday, June 3, 2009==

Romina Clemente and I are trying to find suitable reporter proteins to use. Yesterday, Leland Taylor and Alyndria Thompson were working on ways to insert the gene sequences into the plasmid. Upon seeing how they wanted to manipulated the reporter gene to include the logical clauses, we came up with a few criteria for the reporter genes we would use. The following criteria for genes are listed in order of the broadest aspect to look at to the narrowest aspect:
a) Doesn't contain restriction sites for the 4 restriction enzymes (EcoR1, Xbal, Spel, Pst1) used to cleave the Biobrick part out of the plasmid.
b) Contains 6 cutter restriction sites.
c) These restriction enzymes aren't blunt (cleave straight down at one spot).
d) These restriction sites are close to thge 5' (beginning) end of the sequence.
e) These enzymes are easiest to work with and cheapest.
We are finding the part numbers of the reporter genes we want to use (antibiotic resistance, fluorescence, LacZ) through our own GCAT because we know these ones work. We are then locating these parts on the parts registry [1] website. We copied and pasted the gene sequences we obtained from the registry onto the ApE software [2]. From here, we were able to generate a genetic map of each gene that outlined each restriction site that fit our criteria. We put each genetic map, alongside the part number used, into a Word document.

http://gcat.davidson.edu/GcatWiki/images/0/0e/Restriction_Site_Mapping_on_Reporter_Genes.doc

Later on in the day, we decided that the best way to test for suppression would involve placing the 5mers at the beginning of the reporter gene instead of inserting them into the reporter gene. To do this, we decided that the gene along with its start codon need to be expressed after the 5mer. We will insert a BioBrick plasmid with a 5mer and start codon that will be incorporated before the gene:
RBS-6-8nt-ATG-5mer--ATG-gene

Leland Taylor assembled the oligos that we will need for the BioBrick pieces.

In order to test whether the reporter protein would be expressed regardless of the 5mer being suppressed or not, we would need to remove the suppressor tRNAs from the cell.

Meanwhile, I helped figure out the coding sequence that we will need for the suppressor tRNAs that will bind to the 5mers. We decided that we would be using the following suppressor tRNAs on the Davidson side: CUAGU, CCCUC, CGGUC, CCAUC, and CCACU. We searched through several papers, with little result and finally emailed Dr. Christopher Anderson of UC Berkeley to request the DNA sequence. He emailed us back with a generic DNA code that we could use to create the tRNA; however, for each suppressor tRNA we would use, we would need to change base pairs in the anticodon loop.

The length of the tRNA is 92 nt. The anticodon loop is comprised of 9 nt. Once we have BioBricked the DNA sequence with 4 restriction sites and supplemental nucleotides, the length of the entire gene is 144 nucleotides. This is made up of the 92 nt +22 nt on each side of the sequence (44) + 4 nt on each side of the restriction ends (8).

At the end of the day, we decided to use tetracycline resistance and RFP as our reporter proteins for this tester experiment.

==Thursday, June 4, 2009==

This morning, I began the day by learning how to use the PEARL prgram. Olivia Ho-Shing sat with me and walked me through the program script. And then, Leland Taylor and Shashank Suresh had a problem they wanted us to solve. Leland explained that we need to make sure there is a stop codon in the gene if we fail to suppress the 5mer. The stop codon needs to be before we find another 5mer. They also wanted us to run through both the RFP and Tet Resistance genes to make sure that they did not contain any of the anticodon sequences from our 5 frameshift suppressor tRNAs so as to throw the translation out of frame. They further wanted us to confirm that if we do have successful suppression, there are no stop codons in the middle of the RFP and Tet Resistance genes. PEARL did not find any matches for the suppressor tRNAs within genes. We found 3 matches for stop codons UAA. Two of these occured at the end of the Tet and one at the end of RFP, where they should normally be to end translation into a protein.

Following this, Olivia Ho-Shing and I tried to determine how we can add BioBrick ends to our single stranded DNA sequences that code for the suppressor tRNAs. Our ultimate goal is to find complements for these single strands so they can be put into plasmids. We found the standard prefixes and suffixes for sequences that do not contain ATG (becuase this sequence is a functional RNA and will not be translated) and we placed these before and after the altered 92bp sequence that Dr. Anderson provided us with. The prefixes and suffixes are necessary because they are the extra nucleotides that will be mimicking a "restriction enzyme cutting site". The single strand appeared as below after the Bio Brick prefixes and suffixes were added:

22ntPREFIX--32nt--VARIABLE ANTICODON LOOP--51nt--21ntSUFFIX

We used the suppressor codon CUAGU as our example and placed its appropriate anticodon loop in the sequence. We put this into the lancelator that would give us the other strand as well as the oligos we would need for ideal construction of this double stranded DNA fragment to put into the plasmid. We recieved a total of 4 oligos, 2 of which were variable and 2 of which were constant for all 6 different suppressor tRNAs. Using this double strand, we simulated a restriction enzyme digestion by EcoRI and PstI. We took off the G at both ends of the top strand and then removed the first five nucleotides on each end of the bottom strand. This made the top strand longer than the bottom one. We then proceeded to take the two variable strands and changed the anticodon loop nt for each suppressor tRNA we used.

Around lunchtime, we encountered a problem with the BioBrick scar in the "ATG-5mer subpiece" of the test project. It turns out that after annealing the sticky ends of the Xba1 and Spe1 sites, the scar created had a stop codon TAG (UAG) that was in frame with the suppressed codon. When the Xba1 site on insert anneals to the Spe1 site on the plasmid (the green portions), we get a scar that reads TACTAG.
[[Image:screen-capture.png]]

For the afternoon, Olivia Ho-Shing and I finished assembly of the oligos of the suppressor tRNA codes. We assembled the document below:

http://gcat.davidson.edu/GcatWiki/images/5/5e/TRNAoligostoOrder.doc

We were assigned tasks of creating "cartoons" to illustrate the phenomenon that had occurred. No one could successfully do this however, because we could not understand how the piece had formed. The parts registry website provided wrong information on the restriction site of Spe1 which slowed our efficiency. This project turned into an effort to try to find two other different restriction enzymes that have complementary sticky ends other than Xba1 and Spe1. We want to get rid oc the scar that contains TACTAG.

We ended up deciding that a hybrid of two ideas was needed: restriction enzymes would need to be used to make several reporter gene sequences with different beginnings dependending on the suppressor tRNA sequence needed.

==Friday, June 5, 2009==

Our goal for this morning was to discuss the hybrid PCR approach to assemble the part that comes before the gene we wanted to express. We decided that we would have a BioBrick prefix and a 5mer followed by a sequence of nucleotides whose identity was up to us. Using PCR we would get the second double strand if we have the following:

BioBrick Prefix--ATG--5mer--gene until we get to restriction site

The PCR primer would attach to the ATG and the 5mer and would replicate the complementary base pairs to make a double stranded DNA sequence. As several of us were confused about PCR methods, we looked at the online link provided by Alyndria Thompson's link to the PCR interactive website which was very helpful. We also did some research to discover that the restriction enzyme BseRI (which is contained in the YFP that we wanted to alter) is not compatible with any other restriction enzyme. This is good because we did not want to plasmid containing this gene to close up with Xba1 once we digested it.

The team experienced some confusion with the hybrid idea and spoke to Clif Davis of Missouri Western University on the phone. He told us that there will be one part that is a fragment of the beginning of some nucleotides as a restriction cleaving site, ATG, the logical clause, and the start of the YFP gene that will be placed into a plasmid containing a space. To the left of the space will be the BioBrick Prefix and the right of the space will contain the rest of the genes in the YFP. Therefore, we decided not to alter any BioBrick ends and just look at restriction sites at the beginning of reporter proteins. The small fragment that will be placed into the plasmid will be made by PCR. The PCR will attach a primer onto the ATG and 5mer and replicate.

While we were discussing this, we expanded upon the idea that we can do whatever we want with the "suffix" portion between the 5mer and the beginning of YFP. We decided it would be best to remove ATG and add the first 5 amino acids and the restriction enzyme site. Along with this, if we have the 5mer coding for a certain amino acid (ie methionine) we could replace another codon that codes for methionine WITH that 5mer so long as this codon is contained within the first 15 nt. This way, we would not be altering the protein.

Olivia Ho-Shing came up with an idea of cascading PCR effect for longer logical clauses. Post translational modification of the protein was another one of her ideas. This applies because although we may code for the protein in a given way, the protein may modify or fix itself up to perform a slightly different function in its tertiary form.

Dr. Campbell suggested that we can look up restriction enzymes 400-500 base pairs into the reporter protein we would like to use; however, we have more flexibility in our procedure if we choose more "hardy" cutters. So instead, we could cut with EcoRI instead of with Xba1.

During the afternoon, we set about three tasks:
1) Which primers to use
2) Which promoters to use
3) Which restriction enzymes and how they will determine use of reporter proteins

Olivia Ho-Shing and I began looking up which restriction enzymes were strong so we could base our reporters on that. We began with Tet and RFP since those were the two reporters we had started out with choosing. I found a website that provided enzyme cleavage efficiency based upon the proximity of the site to the end of the PCR fragment that contains it. We discovered the following based upon the attached documents:

http://gcat.davidson.edu/GcatWiki/images/8/8f/Restriction_Enzyme_Cleavage_Efficiency.doc
http://gcat.davidson.edu/GcatWiki/images/9/9a/07Jun09_RE_Cutting_Efficiency_oh.doc

We decided that these two reporters contained efficient enzymes in a good quantity to choose from. On Monday, we will look through these enzymes once more and finalize which enzymes we would like to use with RFP and Tet.

==Sunday, June 7, 2009==

We did preparations for the mini prep this evening. The following notes are courtesy of Olivia Ho-Shing.

1. Sterically transfer ~2mL LB+AMP (AMP are ampicillin resistant plasmids) *Never put fluid back into bottle once it has entered pipette*
2. Use forceps to grab toothpick, scrap some frozen cells, rub in tube with top

'''Tubes'''

S03511 (2)

K091111

B0030

I715039

K091112

I715039

J31007 (2)

B0034

E1010

S03710

==Monday, June 8, 2009==

We reviewed the PCR process this morning as well as the restriction enzyme cleavage process.

Then, Leland Taylor, Olivia Ho-Shing and I all worked on converting the tRNA oligos we made to the template that Missouri Western used. The oligos we had previously made were different from the ones that Dr. Anderson had originally emailed us because further communication between Dr. Anderson and Missouri Western resulted in a better template.

We tried several times to minimize the number of oligos while keeping their individual lengths under 80bp. We managed to do this in the end by creating 7 oligos, 2 of which are variable between the suppressor tRNAs.
We reviewed the following document and finalized the oligos that we needed to order for the frameshift suppressor tRNAs. We decided to keep both the 10bp and 9bp anticodons of CCAUC.

I also looked up the amino acids that corresponded to the FS (frameshift suppressor) tRNAs for future use:

1. CUAGU = Leu
2. CCCUC = Pro
3. CGGUC = Arg
4. CCACU = Pro
5. CCAUC* = Pro
*9 and 10 bp anticodons

Also, we have decided to use NcoI as the restriction enzyme for RFP. This cleavage site occurs 419 bp into the gene. We have decided to use BamHI as the enzyme for Tetracycline Resistance and the cleavage site occurs 290bp into the gene. Both of these enzymes are very powerful and hardy.

We also looked over Missouri Western's tRNA oligos and primer oligos which we approved.
Our oligos were also approved by Missouri Western and Dr. Campbell ordered them while we supervised.

In the afternoon we did the miniprep. The mini prep directions were as appear in the link below.

http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_MiniPrep.html

We then worked with the Nano Prep to dilute our solution of DNA to an absorption of 1.5 and then we made labels for the tubes. The chart below shows relevant information we obtained from the NanoDrop for each of the reporter plasmids.

[[Image: Screen-capture-6.png]]

==Tuesday, June 9, 2009==

'''Preparations for restriction enzymes digestion of plasmids'''

Purpose: To determine the size of the plasmid inserts and ensure that plasmids are what we extracted from the cells during the mini-prep.

Things we need:
DNA ~3uL
Buffer 10x (Want to dilute to 1x) so add 2uL
Enzyme 1 ~1uL
Enzme 2 ~1uL
Water ~13uL
Final Volunter about 20 uL.

Need to choose the right buffer for the restriction enzymes. All buffers come in 10x (10 times more concentrated than what you want it to be when you use it). [http://gcat.davidson.edu/GcatWiki/index.php/Davidson_Missouri_W/Double_Digest_Guide Buffer Website]

Total volume of enzymes has to be 10% or less than final volume (most volume 2uL). Barely submerge the pipet tip into the solution when pipeting the enzymes.
Star (*) activity: Restriction enzyme stops cutting specifically and starts cutting everywhere.

DNA can't be seen by itself so we add EtBr (the bigger the insert, the more will bind) so bigger strands appear brighter. samples.

The order of entry doesn't matter, but make sure to enter the enzymes last.

Will have lots of labeled tubes and will put 3uL of appropriate plasmids into the tubes.
In the meantime, we'll make a "Master Mix" of all the other ingredients in a bigger tube. Always make enough volume for one extra tube.

Enzymes usually have an optimal temperature of 37C. We will let this reaction go for an hour and then we will run it on a gel.

'''Gel Electrophoresis Protocol'''

Website online for ideal gel concentration for resolving different sizes of molecules [http://gcat.davidson.edu/iGEM08/gelwebsite/gelwebsite.html Gel Concentration Website].
See protocol on the Davidson/Missouri Western Wookie for running molecules in the gel and how to digest with restriction enzymes.

'''What's Next?'''

1. 5' end additions
a. Using PCR
b. Cut at the restriction sites EcoR1 and either Bam H1 or Nco1
c. Cut plasmid and reporter with same restriction enzymes as above
d. Run a gel and purify "keeper" DNA (Ligate transform, screen, sequence)
e. Then, we take the "keeper" DNA and combine it with the insert from part

We can have steps d and e ready in advance.

2. tRNAs
a. Assemble the oligos into genes (Requires lots of calculations beforehand, but the actual steps )
b. Plasmids cut up with EcoR1 and Pst1 '''WE CAN USE THE "WASTE" PLASMID FROM THE RESTRICTION ENZYME DIGEST'''
c. Gel purify the plasmids (Ligate transform, screen, sequence)

The following tables include Master Mix measurements, agarose gel concentrations and buffers.
http://gcat.davidson.edu/GcatWiki/images/0/08/Shamitapic3.png
http://gcat.davidson.edu/GcatWiki/images/d/d1/Shamitapic4.png
http://gcat.davidson.edu/GcatWiki/images/6/6f/Shamitapic5.png

We made the Master Mix with our DNA and put it in the incubator at 10:40 AM.

'''Preparing the Gels'''

Now, we will be making 2 gels, 1 at 0.8% and 2.5% (weight to volume percent). The gel volume is 60mL.

'''Definition of 1% solution:
1 gram of agarose/ 100mL buffer'''

So for a 0.8% solution: 0.8 grams agarose/100mL buffer AND we need 60mL (fixed volume for casts run) so multiply that by 0.6.
Final concentration:

.48 grams agarose/60mL buffer

1 uL of EtBr stock for 60mL of gel.
EtBr is in the buffer so it is constantly replenishing itself even though it is moving toward one end in order to make bands on the other end visible.

After Dr. Campbell's demonstration, we each helped prepare a 0.8% buffer solution using the agarose powder, buffer and EtBr. We mixed together the first two substances in a 200mL Erlenmeyer flask and heated it for 120 seconds in the microwave. We then added EtBr and poured the gel solution into the gel cast.

'''Agar Plate'''

After lunch, we grew some more cells for each of the 11 parts in an agar plate labeled "LBAMP-AMC-9 June" so that so would have extra cells that could be stored for up to 2 weeks.
We made small circles on the agar plate and numbered them 1-11. The numbers corresponded to the following parts. We put the plate in the incubator overnight to let the cells grow.

1. B0030 AMC
2. K091111
3. S03710
4. I715039 OEH
5. S03511 ORH
6. I715039 AST
7. B0030 SDP
8. K091112
9. J31007 LJH
10. J31007 REC
11. S03511

'''Unloading the Gels'''

The following shows which parts we entered in which gels:

[[Image: screen-capture-2.png]]

We unloaded the 2.5% agarose first and after observing the bands under a UV light, put the gel into the BioRad machine to observe the bands. The image below is of a Molecular Weight Marker that shows the number of bp in control fragments of DNA according to the distance they travel down the gel.

[[Image: screen-capture-1.png]]

We obtained the following printout for the first gel that was 2.5% Agarose.
[[Image: 2009-06-09_14hr_44min.jpg]]

The bands in the first well at the bottom are those of the molecular weight marker. We skipped the lane above (to the right) of the MWM and entered the rest of the inserts as described in the above table. A light band is visible for each of the 4 other wells. In Lanes 3 and 4 we concluded we are supposed to have approximately 45 bp because the insert is 15bp + 30 bp on either side of the insert (Xba1 and Spe1 restrictions sites + sticky ends + extra bases). We used the same logic for Lanes 5 and 6 which are supposed to contain approximately 86 base pairs.

One discrepancy in this gel, however, is that Lanes 5 and 6, which according to their labels respectively contain the LacIQ promoter and the Lac IQ1 promoter. According to the information online at the parts registry website, pLacIQ1 should be the same length as pLacIQ. The gels do not show this, but instead show that the sample in Lane 5 is smaller than that in Lane 6 because Lane 5 traveled further. Leland Taylor and Romina Clemente sent out an email to Pallavi Penumetcha to ask about this because she had written a paper about this.

We then unloaded the 0.8% agarose gel and then placed this in the BioRad machine under the same protocol as above. The following image is the 0.8% Agarose.

[[Image:2009-06-09_15hr_21min.jpg]]

We observed that the bands in Lane 1 (which is now at the top) are examples of high intensity bands. The plasmids contained in the original well are twice the intensity of the insert.

In Lane 5, however, we observed several different bands. This could be due to the extra DNA that was sitting on the top of the well. We decided to re-expose the gel to get better observations on Lanes 3 and 4 because the bands were not as intense. We anticipated seeing a band at about 250 bp because this was the size of the insert (plus extra bp). After re-exposing the gel, we did not find any difference and still did not see any bands in these lanes. We have decided to redo the gel on the part S03511 and so we prepared the master mix with that DNA and incubated it so that we could run the gel tomorrow.

We also cut out the "empty" plasmids from the gel so that we could use this as vectors for the coding sequence of the suppressor tRNAs.

Tomorrow we will be planning out specific tasks for people once the oligos arrive.

==Wednesday, June 10, 2009==

We began this morning by making the agarose gel with the same protocol as yesterday. This time, since we are just re-doing both S03511 inserts, we will have a 1.8% agarose gel.

'''OUTLINE: WHERE DO WE STAND?'''

Yesterday
a. Isolated empty plasmids
b. Verified most inserts
c. Waiting for:
>tRNA oligos
>Primers
a. Will amplify the reporter gene in the plasmid itself

Next?
a. When Primers Arrive
> Amplify the reporter genes with primers (PCR takes ~3 hours)
> Calculate how much reporter plasmid we will need (Start with 50x more than we actually will need)
> Cut amplified sequence (EcoR1 and BamH1/Nco1) then purify what we need
> Cut reporter plasmid (EcoR1 and BamH1/Nco1) then purify what we need
> Ligate amplified sequence and reporter plasmid
b. When tRNA oligos arrive
> Calculate how much of each oligo we will need (takes a while...)
> "Boil and cool" tRNA oligos together
> Ligate fragments (dsDNA) into plasmids we isolated yesterday (~5 minutes)
> Transform the plasmids (~20 minutes)

* LOOK THROUGH LIGATION PROTOCOL (How to assemble genes)
* LOOK THROUGH TRANSFORMATION PROTOCOL

''What we need to get done today'':
1. Prepare the control for the Tet Resistance by BioBricking the part pLac+RBS (S03511) with the TetA (J31007).
2. Check the part number for RFP and also for pLac+RBS+RFP
3. Read through ligation protocol
4. See how many nanograms of plasmid we will need for the tRNAs and for the inserts and multiply this number by 50.
> Check if we have enough plasmids from our mini-prep
5. Purify the plasmids from the gel

After we made this outline, Leland, Olivia and I analyzed the gel we ran this morning and did not see a more than one band in the 2 lanes that contained DNA from S03511 (image below). Since we did not see multiple bands in yesterday's gel either, we conclude that there is something wrong with the DNA rather than with the restriction enzymes that we used to cut it. We concluded that there is probably not an insert in this plasmid both the part and the plasmid are useless.

[[Image: 2009-06-10 10hr 30min.jpg]]

Now, we do not have an immediately accessible promoter. We have decided to order the pBAD promoter from Missouri Western and we will attach the RBS+Tet to this plasmid. Our insert for this will be the larger gene because it is easier to manipulate in the gel than the small gene.

Alyndria Thompson and Romina Clemente are figuring out the size of the plasmids including the reporter genes.

After lunch we performed several steps to isolate and purify the gel to get the empty plasmids will be the vectors for the tRNA DNA. Then, we used the NanoDrop to assess the purity of our DNA. We found that the plasmids were more pure than we had expected. The following values were obtained:

Abs 1.850
A-260 10 mm path 0.172
A-280 10 mm path 0.075
260/280 2.28
260/230 0.09

We then followed the protocol for preparing the agar plates. We put the plate with the agar solution in the Autoclave.

While the solution was in the autoclave, we discussed that we don't really need a promoter in front of RBS+Tet for the gene to be expressed because Tet expression is so strong that the gene would translate without a promoter. However, it would be a good idea to have the promoter because if it was possible to reinforce the translation of the reporter protein, we wanted to do that.

We found that there were some tubes in the lab fridge that contained the pBAD and pLac promoters with RBS. We decided to use these as the promoter and RBS for our experiments. The pLac promoter will be used for RFP and the pBad promoter used with Tet Resistance. We decided to use the pBad with Tet because it is weaker in comparison to pLac. As expression of the Tet gene can be toxic to a cell, we do not want to overexpress the gene with a stronger promoter.

Once the agar solution was sterilized in the Autoclave, we removed it, cooled it, and pipetted the antibiotic resistance into it. We then poured the solution into the plates, removed any bubbles, and let the gel form.

Alyndria Thompson and Romina Clemente concluded that we had more than enough recieving vectors for both the RFP and Tetracycline genes. Since we need 50 nanograms, it is wise to have at least 50 times that much that we can work with, so we need at least 2,500 ng.

RFP 1: 77.6 x 4 = 310.4 ng/uL
x 20 uL = 3,272 ng
RFP 2: 59.5 x 4 = 238 ng/uL
x 20 uL = 4,760 ng
Tet: 40.9 x 4 = 163.6 ng/uL
x 20 uL = 3,272 ng

According to these calculations we have more than 50 times what we need for both instances of RFP and Tet.

Tomorrow, we can prepare the Master Mix for the digestions of both reporter plamids and then add the restriction enzymes once they arrive. Then, we can follow through with the rest of the plans we had made above in the section titles "Next?".

==Thursday, June 11, 2009==

We began this morning by calculating how many uL we would need for each of the mixes for digesting our reporter plasmids. We decided to double the final volume of each mix to 40uL instead of 20uL. Since we knew the concentration of each plasmid and also knew we needed 2500 ng, we cross multiplied to find the uL of DNA we would need for each.

Concentration Calculations:
310.4 ng/1 uL = 2500ng/x uL
x = 8.05 uL

Here is a chart indicating how much of each we used for the 2 mixes.

[[Image: DoubledigestRFPTetR.png]]

On the Promega website, we found a list of compatible buffers for double digestions. Buffers E and H are compatible for the Tet plasmid and RFP plasmid digestions respectively. We fortunately have both buffers in stock.

[[Image:BamEcoR1.png]] [[Image:NcoEcoR1.png]]

We waited for our waited for the restriction enzymes to arrive and in the meantime, we transformed the plasmids with pLab+RBS, pBAd, and pBad+RBS into ''E.coli'' cells. We want the ''E.coli'' cells to replicate the plasmids with these genes because we have a limited amount of genes. [http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_Transformation.html Zippy Plasmid Transformation]

Once our oligos and restriction enzymes arrived, we got to work right away!

'''PCR (Polymerase Chain Reaction)'''

We calculated how much plasmid we will need for the PCR. We decided to dilute the concentration of our plasmids a hundred fold so that we could get pipetable amounts.

''Change in Concentrations''
310.4 ng/uL x 1/100 (dilution factor) = 3.104 ng/uL (RFP)
163.6 ng/uL x 1/100 (dilution factor) = 0.164 ng/uL (Tet)

''Example shown with RFP''
3.104 ng/1 uL = 1ng/ x uL, x = .32
For Tet, x = .61

We also calculated how much of each primer we would need with the following equation:

C1V1 =C2V2
(100 uM) (x) = (1 uM)( 100 uL)
x= 1 uL

[[Image: screen-capture-8.png]]

Each of the 5 of us made had 2 tubes (one for each reporter protein) because there were 5 different forward primers.To each tube, we added the template DNA (either RFP or Tet), water, the respective reverse primer (either RFP or Tet), and the forward primer depending on which 5mer we had chosen. My 5mer was CCAUC. Finally, we added the Master Mix last because this contained the enzyme (DNA polymerase) that would amplify our desired sequence.

[[Image: Pcrtubes.png]]

We followed the temperature cycles so that PCR could occur. The annealing temperature needed to be 5C less than the smallest melting temperature of the primers. Since the reverse primer of RFP had a melting temp of 66C, we set the temperature of the 3rd cycle on 61C.

[[Image: Temperaturecycle.png]]

'''tRNA Oligo Assembly'''

We needed to calculate the concentration of each of the oligo before carrying out the assembly. The stock concentration of each of the oligo was 100 uM. Since we wanted to have 5uM of the oligo in the test tube, and we know that final volume of the test tube is 20 uL, we just cross multiplied to find the amount we needed to add to the tubes to get this concentration.

(100uM)x = (5uM)(20uL)
x = 1 uL

We added 1 uL of each oligo to the tubes.

We had six total tRNAs we needed to assemble so each of us took one FML (one person took two) and to each tube we added 1uL of each oligo for a total of 7uL, 2uL of buffer, and 11uL of water for a total volume of 20 uL. Five of the oligos were constant and 2 varied for each of the different tRNAs. So everyone pipetted the same 5 oligos and everyone also had 2 unique oligos to add. When all of the oligos were added and the tubes ready, we boiled the tubes at 50C for 5 minutes. We then allowed the tubes to remain in the water to cool very slowly overnight so that the annealing process could take place.

IGEM Notebook

2009-06-12T01:34:44Z

ShPunjabi: /* Thursday, June 11, 2009 */

==Wednesday, June 3, 2009==

Romina Clemente and I are trying to find suitable reporter proteins to use. Yesterday, Leland Taylor and Alyndria Thompson were working on ways to insert the gene sequences into the plasmid. Upon seeing how they wanted to manipulated the reporter gene to include the logical clauses, we came up with a few criteria for the reporter genes we would use. The following criteria for genes are listed in order of the broadest aspect to look at to the narrowest aspect:
a) Doesn't contain restriction sites for the 4 restriction enzymes (EcoR1, Xbal, Spel, Pst1) used to cleave the Biobrick part out of the plasmid.
b) Contains 6 cutter restriction sites.
c) These restriction enzymes aren't blunt (cleave straight down at one spot).
d) These restriction sites are close to thge 5' (beginning) end of the sequence.
e) These enzymes are easiest to work with and cheapest.
We are finding the part numbers of the reporter genes we want to use (antibiotic resistance, fluorescence, LacZ) through our own GCAT because we know these ones work. We are then locating these parts on the parts registry [1] website. We copied and pasted the gene sequences we obtained from the registry onto the ApE software [2]. From here, we were able to generate a genetic map of each gene that outlined each restriction site that fit our criteria. We put each genetic map, alongside the part number used, into a Word document.

http://gcat.davidson.edu/GcatWiki/images/0/0e/Restriction_Site_Mapping_on_Reporter_Genes.doc

Later on in the day, we decided that the best way to test for suppression would involve placing the 5mers at the beginning of the reporter gene instead of inserting them into the reporter gene. To do this, we decided that the gene along with its start codon need to be expressed after the 5mer. We will insert a BioBrick plasmid with a 5mer and start codon that will be incorporated before the gene:
RBS-6-8nt-ATG-5mer--ATG-gene

Leland Taylor assembled the oligos that we will need for the BioBrick pieces.

In order to test whether the reporter protein would be expressed regardless of the 5mer being suppressed or not, we would need to remove the suppressor tRNAs from the cell.

Meanwhile, I helped figure out the coding sequence that we will need for the suppressor tRNAs that will bind to the 5mers. We decided that we would be using the following suppressor tRNAs on the Davidson side: CUAGU, CCCUC, CGGUC, CCAUC, and CCACU. We searched through several papers, with little result and finally emailed Dr. Christopher Anderson of UC Berkeley to request the DNA sequence. He emailed us back with a generic DNA code that we could use to create the tRNA; however, for each suppressor tRNA we would use, we would need to change base pairs in the anticodon loop.

The length of the tRNA is 92 nt. The anticodon loop is comprised of 9 nt. Once we have BioBricked the DNA sequence with 4 restriction sites and supplemental nucleotides, the length of the entire gene is 144 nucleotides. This is made up of the 92 nt +22 nt on each side of the sequence (44) + 4 nt on each side of the restriction ends (8).

At the end of the day, we decided to use tetracycline resistance and RFP as our reporter proteins for this tester experiment.

==Thursday, June 4, 2009==

This morning, I began the day by learning how to use the PEARL prgram. Olivia Ho-Shing sat with me and walked me through the program script. And then, Leland Taylor and Shashank Suresh had a problem they wanted us to solve. Leland explained that we need to make sure there is a stop codon in the gene if we fail to suppress the 5mer. The stop codon needs to be before we find another 5mer. They also wanted us to run through both the RFP and Tet Resistance genes to make sure that they did not contain any of the anticodon sequences from our 5 frameshift suppressor tRNAs so as to throw the translation out of frame. They further wanted us to confirm that if we do have successful suppression, there are no stop codons in the middle of the RFP and Tet Resistance genes. PEARL did not find any matches for the suppressor tRNAs within genes. We found 3 matches for stop codons UAA. Two of these occured at the end of the Tet and one at the end of RFP, where they should normally be to end translation into a protein.

Following this, Olivia Ho-Shing and I tried to determine how we can add BioBrick ends to our single stranded DNA sequences that code for the suppressor tRNAs. Our ultimate goal is to find complements for these single strands so they can be put into plasmids. We found the standard prefixes and suffixes for sequences that do not contain ATG (becuase this sequence is a functional RNA and will not be translated) and we placed these before and after the altered 92bp sequence that Dr. Anderson provided us with. The prefixes and suffixes are necessary because they are the extra nucleotides that will be mimicking a "restriction enzyme cutting site". The single strand appeared as below after the Bio Brick prefixes and suffixes were added:

22ntPREFIX--32nt--VARIABLE ANTICODON LOOP--51nt--21ntSUFFIX

We used the suppressor codon CUAGU as our example and placed its appropriate anticodon loop in the sequence. We put this into the lancelator that would give us the other strand as well as the oligos we would need for ideal construction of this double stranded DNA fragment to put into the plasmid. We recieved a total of 4 oligos, 2 of which were variable and 2 of which were constant for all 6 different suppressor tRNAs. Using this double strand, we simulated a restriction enzyme digestion by EcoRI and PstI. We took off the G at both ends of the top strand and then removed the first five nucleotides on each end of the bottom strand. This made the top strand longer than the bottom one. We then proceeded to take the two variable strands and changed the anticodon loop nt for each suppressor tRNA we used.

Around lunchtime, we encountered a problem with the BioBrick scar in the "ATG-5mer subpiece" of the test project. It turns out that after annealing the sticky ends of the Xba1 and Spe1 sites, the scar created had a stop codon TAG (UAG) that was in frame with the suppressed codon. When the Xba1 site on insert anneals to the Spe1 site on the plasmid (the green portions), we get a scar that reads TACTAG.
[[Image:screen-capture.png]]

For the afternoon, Olivia Ho-Shing and I finished assembly of the oligos of the suppressor tRNA codes. We assembled the document below:

http://gcat.davidson.edu/GcatWiki/images/5/5e/TRNAoligostoOrder.doc

We were assigned tasks of creating "cartoons" to illustrate the phenomenon that had occurred. No one could successfully do this however, because we could not understand how the piece had formed. The parts registry website provided wrong information on the restriction site of Spe1 which slowed our efficiency. This project turned into an effort to try to find two other different restriction enzymes that have complementary sticky ends other than Xba1 and Spe1. We want to get rid oc the scar that contains TACTAG.

We ended up deciding that a hybrid of two ideas was needed: restriction enzymes would need to be used to make several reporter gene sequences with different beginnings dependending on the suppressor tRNA sequence needed.

==Friday, June 5, 2009==

Our goal for this morning was to discuss the hybrid PCR approach to assemble the part that comes before the gene we wanted to express. We decided that we would have a BioBrick prefix and a 5mer followed by a sequence of nucleotides whose identity was up to us. Using PCR we would get the second double strand if we have the following:

BioBrick Prefix--ATG--5mer--gene until we get to restriction site

The PCR primer would attach to the ATG and the 5mer and would replicate the complementary base pairs to make a double stranded DNA sequence. As several of us were confused about PCR methods, we looked at the online link provided by Alyndria Thompson's link to the PCR interactive website which was very helpful. We also did some research to discover that the restriction enzyme BseRI (which is contained in the YFP that we wanted to alter) is not compatible with any other restriction enzyme. This is good because we did not want to plasmid containing this gene to close up with Xba1 once we digested it.

The team experienced some confusion with the hybrid idea and spoke to Clif Davis of Missouri Western University on the phone. He told us that there will be one part that is a fragment of the beginning of some nucleotides as a restriction cleaving site, ATG, the logical clause, and the start of the YFP gene that will be placed into a plasmid containing a space. To the left of the space will be the BioBrick Prefix and the right of the space will contain the rest of the genes in the YFP. Therefore, we decided not to alter any BioBrick ends and just look at restriction sites at the beginning of reporter proteins. The small fragment that will be placed into the plasmid will be made by PCR. The PCR will attach a primer onto the ATG and 5mer and replicate.

While we were discussing this, we expanded upon the idea that we can do whatever we want with the "suffix" portion between the 5mer and the beginning of YFP. We decided it would be best to remove ATG and add the first 5 amino acids and the restriction enzyme site. Along with this, if we have the 5mer coding for a certain amino acid (ie methionine) we could replace another codon that codes for methionine WITH that 5mer so long as this codon is contained within the first 15 nt. This way, we would not be altering the protein.

Olivia Ho-Shing came up with an idea of cascading PCR effect for longer logical clauses. Post translational modification of the protein was another one of her ideas. This applies because although we may code for the protein in a given way, the protein may modify or fix itself up to perform a slightly different function in its tertiary form.

Dr. Campbell suggested that we can look up restriction enzymes 400-500 base pairs into the reporter protein we would like to use; however, we have more flexibility in our procedure if we choose more "hardy" cutters. So instead, we could cut with EcoRI instead of with Xba1.

During the afternoon, we set about three tasks:
1) Which primers to use
2) Which promoters to use
3) Which restriction enzymes and how they will determine use of reporter proteins

Olivia Ho-Shing and I began looking up which restriction enzymes were strong so we could base our reporters on that. We began with Tet and RFP since those were the two reporters we had started out with choosing. I found a website that provided enzyme cleavage efficiency based upon the proximity of the site to the end of the PCR fragment that contains it. We discovered the following based upon the attached documents:

http://gcat.davidson.edu/GcatWiki/images/8/8f/Restriction_Enzyme_Cleavage_Efficiency.doc
http://gcat.davidson.edu/GcatWiki/images/9/9a/07Jun09_RE_Cutting_Efficiency_oh.doc

We decided that these two reporters contained efficient enzymes in a good quantity to choose from. On Monday, we will look through these enzymes once more and finalize which enzymes we would like to use with RFP and Tet.

==Sunday, June 7, 2009==

We did preparations for the mini prep this evening. The following notes are courtesy of Olivia Ho-Shing.

1. Sterically transfer ~2mL LB+AMP (AMP are ampicillin resistant plasmids) *Never put fluid back into bottle once it has entered pipette*
2. Use forceps to grab toothpick, scrap some frozen cells, rub in tube with top

'''Tubes'''

S03511 (2)

K091111

B0030

I715039

K091112

I715039

J31007 (2)

B0034

E1010

S03710

==Monday, June 8, 2009==

We reviewed the PCR process this morning as well as the restriction enzyme cleavage process.

Then, Leland Taylor, Olivia Ho-Shing and I all worked on converting the tRNA oligos we made to the template that Missouri Western used. The oligos we had previously made were different from the ones that Dr. Anderson had originally emailed us because further communication between Dr. Anderson and Missouri Western resulted in a better template.

We tried several times to minimize the number of oligos while keeping their individual lengths under 80bp. We managed to do this in the end by creating 7 oligos, 2 of which are variable between the suppressor tRNAs.
We reviewed the following document and finalized the oligos that we needed to order for the frameshift suppressor tRNAs. We decided to keep both the 10bp and 9bp anticodons of CCAUC.

I also looked up the amino acids that corresponded to the FS (frameshift suppressor) tRNAs for future use:

1. CUAGU = Leu
2. CCCUC = Pro
3. CGGUC = Arg
4. CCACU = Pro
5. CCAUC* = Pro
*9 and 10 bp anticodons

Also, we have decided to use NcoI as the restriction enzyme for RFP. This cleavage site occurs 419 bp into the gene. We have decided to use BamHI as the enzyme for Tetracycline Resistance and the cleavage site occurs 290bp into the gene. Both of these enzymes are very powerful and hardy.

We also looked over Missouri Western's tRNA oligos and primer oligos which we approved.
Our oligos were also approved by Missouri Western and Dr. Campbell ordered them while we supervised.

In the afternoon we did the miniprep. The mini prep directions were as appear in the link below.

http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_MiniPrep.html

We then worked with the Nano Prep to dilute our solution of DNA to an absorption of 1.5 and then we made labels for the tubes. The chart below shows relevant information we obtained from the NanoDrop for each of the reporter plasmids.

[[Image: Screen-capture-6.png]]

==Tuesday, June 9, 2009==

'''Preparations for restriction enzymes digestion of plasmids'''

Purpose: To determine the size of the plasmid inserts and ensure that plasmids are what we extracted from the cells during the mini-prep.

Things we need:
DNA ~3uL
Buffer 10x (Want to dilute to 1x) so add 2uL
Enzyme 1 ~1uL
Enzme 2 ~1uL
Water ~13uL
Final Volunter about 20 uL.

Need to choose the right buffer for the restriction enzymes. All buffers come in 10x (10 times more concentrated than what you want it to be when you use it). [http://gcat.davidson.edu/GcatWiki/index.php/Davidson_Missouri_W/Double_Digest_Guide Buffer Website]

Total volume of enzymes has to be 10% or less than final volume (most volume 2uL). Barely submerge the pipet tip into the solution when pipeting the enzymes.
Star (*) activity: Restriction enzyme stops cutting specifically and starts cutting everywhere.

DNA can't be seen by itself so we add EtBr (the bigger the insert, the more will bind) so bigger strands appear brighter. samples.

The order of entry doesn't matter, but make sure to enter the enzymes last.

Will have lots of labeled tubes and will put 3uL of appropriate plasmids into the tubes.
In the meantime, we'll make a "Master Mix" of all the other ingredients in a bigger tube. Always make enough volume for one extra tube.

Enzymes usually have an optimal temperature of 37C. We will let this reaction go for an hour and then we will run it on a gel.

'''Gel Electrophoresis Protocol'''

Website online for ideal gel concentration for resolving different sizes of molecules [http://gcat.davidson.edu/iGEM08/gelwebsite/gelwebsite.html Gel Concentration Website].
See protocol on the Davidson/Missouri Western Wookie for running molecules in the gel and how to digest with restriction enzymes.

'''What's Next?'''

1. 5' end additions
a. Using PCR
b. Cut at the restriction sites EcoR1 and either Bam H1 or Nco1
c. Cut plasmid and reporter with same restriction enzymes as above
d. Run a gel and purify "keeper" DNA (Ligate transform, screen, sequence)
e. Then, we take the "keeper" DNA and combine it with the insert from part

We can have steps d and e ready in advance.

2. tRNAs
a. Assemble the oligos into genes (Requires lots of calculations beforehand, but the actual steps )
b. Plasmids cut up with EcoR1 and Pst1 '''WE CAN USE THE "WASTE" PLASMID FROM THE RESTRICTION ENZYME DIGEST'''
c. Gel purify the plasmids (Ligate transform, screen, sequence)

The following tables include Master Mix measurements, agarose gel concentrations and buffers.
http://gcat.davidson.edu/GcatWiki/images/0/08/Shamitapic3.png
http://gcat.davidson.edu/GcatWiki/images/d/d1/Shamitapic4.png
http://gcat.davidson.edu/GcatWiki/images/6/6f/Shamitapic5.png

We made the Master Mix with our DNA and put it in the incubator at 10:40 AM.

'''Preparing the Gels'''

Now, we will be making 2 gels, 1 at 0.8% and 2.5% (weight to volume percent). The gel volume is 60mL.

'''Definition of 1% solution:
1 gram of agarose/ 100mL buffer'''

So for a 0.8% solution: 0.8 grams agarose/100mL buffer AND we need 60mL (fixed volume for casts run) so multiply that by 0.6.
Final concentration:

.48 grams agarose/60mL buffer

1 uL of EtBr stock for 60mL of gel.
EtBr is in the buffer so it is constantly replenishing itself even though it is moving toward one end in order to make bands on the other end visible.

After Dr. Campbell's demonstration, we each helped prepare a 0.8% buffer solution using the agarose powder, buffer and EtBr. We mixed together the first two substances in a 200mL Erlenmeyer flask and heated it for 120 seconds in the microwave. We then added EtBr and poured the gel solution into the gel cast.

'''Agar Plate'''

After lunch, we grew some more cells for each of the 11 parts in an agar plate labeled "LBAMP-AMC-9 June" so that so would have extra cells that could be stored for up to 2 weeks.
We made small circles on the agar plate and numbered them 1-11. The numbers corresponded to the following parts. We put the plate in the incubator overnight to let the cells grow.

1. B0030 AMC
2. K091111
3. S03710
4. I715039 OEH
5. S03511 ORH
6. I715039 AST
7. B0030 SDP
8. K091112
9. J31007 LJH
10. J31007 REC
11. S03511

'''Unloading the Gels'''

The following shows which parts we entered in which gels:

[[Image: screen-capture-2.png]]

We unloaded the 2.5% agarose first and after observing the bands under a UV light, put the gel into the BioRad machine to observe the bands. The image below is of a Molecular Weight Marker that shows the number of bp in control fragments of DNA according to the distance they travel down the gel.

[[Image: screen-capture-1.png]]

We obtained the following printout for the first gel that was 2.5% Agarose.
[[Image: 2009-06-09_14hr_44min.jpg]]

The bands in the first well at the bottom are those of the molecular weight marker. We skipped the lane above (to the right) of the MWM and entered the rest of the inserts as described in the above table. A light band is visible for each of the 4 other wells. In Lanes 3 and 4 we concluded we are supposed to have approximately 45 bp because the insert is 15bp + 30 bp on either side of the insert (Xba1 and Spe1 restrictions sites + sticky ends + extra bases). We used the same logic for Lanes 5 and 6 which are supposed to contain approximately 86 base pairs.

One discrepancy in this gel, however, is that Lanes 5 and 6, which according to their labels respectively contain the LacIQ promoter and the Lac IQ1 promoter. According to the information online at the parts registry website, pLacIQ1 should be the same length as pLacIQ. The gels do not show this, but instead show that the sample in Lane 5 is smaller than that in Lane 6 because Lane 5 traveled further. Leland Taylor and Romina Clemente sent out an email to Pallavi Penumetcha to ask about this because she had written a paper about this.

We then unloaded the 0.8% agarose gel and then placed this in the BioRad machine under the same protocol as above. The following image is the 0.8% Agarose.

[[Image:2009-06-09_15hr_21min.jpg]]

We observed that the bands in Lane 1 (which is now at the top) are examples of high intensity bands. The plasmids contained in the original well are twice the intensity of the insert.

In Lane 5, however, we observed several different bands. This could be due to the extra DNA that was sitting on the top of the well. We decided to re-expose the gel to get better observations on Lanes 3 and 4 because the bands were not as intense. We anticipated seeing a band at about 250 bp because this was the size of the insert (plus extra bp). After re-exposing the gel, we did not find any difference and still did not see any bands in these lanes. We have decided to redo the gel on the part S03511 and so we prepared the master mix with that DNA and incubated it so that we could run the gel tomorrow.

We also cut out the "empty" plasmids from the gel so that we could use this as vectors for the coding sequence of the suppressor tRNAs.

Tomorrow we will be planning out specific tasks for people once the oligos arrive.

==Wednesday, June 10, 2009==

We began this morning by making the agarose gel with the same protocol as yesterday. This time, since we are just re-doing both S03511 inserts, we will have a 1.8% agarose gel.

'''OUTLINE: WHERE DO WE STAND?'''

Yesterday
a. Isolated empty plasmids
b. Verified most inserts
c. Waiting for:
>tRNA oligos
>Primers
a. Will amplify the reporter gene in the plasmid itself

Next?
a. When Primers Arrive
> Amplify the reporter genes with primers (PCR takes ~3 hours)
> Calculate how much reporter plasmid we will need (Start with 50x more than we actually will need)
> Cut amplified sequence (EcoR1 and BamH1/Nco1) then purify what we need
> Cut reporter plasmid (EcoR1 and BamH1/Nco1) then purify what we need
> Ligate amplified sequence and reporter plasmid
b. When tRNA oligos arrive
> Calculate how much of each oligo we will need (takes a while...)
> "Boil and cool" tRNA oligos together
> Ligate fragments (dsDNA) into plasmids we isolated yesterday (~5 minutes)
> Transform the plasmids (~20 minutes)

* LOOK THROUGH LIGATION PROTOCOL (How to assemble genes)
* LOOK THROUGH TRANSFORMATION PROTOCOL

''What we need to get done today'':
1. Prepare the control for the Tet Resistance by BioBricking the part pLac+RBS (S03511) with the TetA (J31007).
2. Check the part number for RFP and also for pLac+RBS+RFP
3. Read through ligation protocol
4. See how many nanograms of plasmid we will need for the tRNAs and for the inserts and multiply this number by 50.
> Check if we have enough plasmids from our mini-prep
5. Purify the plasmids from the gel

After we made this outline, Leland, Olivia and I analyzed the gel we ran this morning and did not see a more than one band in the 2 lanes that contained DNA from S03511 (image below). Since we did not see multiple bands in yesterday's gel either, we conclude that there is something wrong with the DNA rather than with the restriction enzymes that we used to cut it. We concluded that there is probably not an insert in this plasmid both the part and the plasmid are useless.

[[Image: 2009-06-10 10hr 30min.jpg]]

Now, we do not have an immediately accessible promoter. We have decided to order the pBAD promoter from Missouri Western and we will attach the RBS+Tet to this plasmid. Our insert for this will be the larger gene because it is easier to manipulate in the gel than the small gene.

Alyndria Thompson and Romina Clemente are figuring out the size of the plasmids including the reporter genes.

After lunch we performed several steps to isolate and purify the gel to get the empty plasmids will be the vectors for the tRNA DNA. Then, we used the NanoDrop to assess the purity of our DNA. We found that the plasmids were more pure than we had expected. The following values were obtained:

Abs 1.850
A-260 10 mm path 0.172
A-280 10 mm path 0.075
260/280 2.28
260/230 0.09

We then followed the protocol for preparing the agar plates. We put the plate with the agar solution in the Autoclave.

While the solution was in the autoclave, we discussed that we don't really need a promoter in front of RBS+Tet for the gene to be expressed because Tet expression is so strong that the gene would translate without a promoter. However, it would be a good idea to have the promoter because if it was possible to reinforce the translation of the reporter protein, we wanted to do that.

We found that there were some tubes in the lab fridge that contained the pBAD and pLac promoters with RBS. We decided to use these as the promoter and RBS for our experiments. The pLac promoter will be used for RFP and the pBad promoter used with Tet Resistance. We decided to use the pBad with Tet because it is weaker in comparison to pLac. As expression of the Tet gene can be toxic to a cell, we do not want to overexpress the gene with a stronger promoter.

Once the agar solution was sterilized in the Autoclave, we removed it, cooled it, and pipetted the antibiotic resistance into it. We then poured the solution into the plates, removed any bubbles, and let the gel form.

Alyndria Thompson and Romina Clemente concluded that we had more than enough recieving vectors for both the RFP and Tetracycline genes. Since we need 50 nanograms, it is wise to have at least 50 times that much that we can work with, so we need at least 2,500 ng.

RFP 1: 77.6 x 4 = 310.4 ng/uL
x 20 uL = 3,272 ng
RFP 2: 59.5 x 4 = 238 ng/uL
x 20 uL = 4,760 ng
Tet: 40.9 x 4 = 163.6 ng/uL
x 20 uL = 3,272 ng

According to these calculations we have more than 50 times what we need for both instances of RFP and Tet.

Tomorrow, we can prepare the Master Mix for the digestions of both reporter plamids and then add the restriction enzymes once they arrive. Then, we can follow through with the rest of the plans we had made above in the section titles "Next?".

==Thursday, June 11, 2009==

We began this morning by calculating how many uL we would need for each of the mixes for digesting our reporter plasmids. We decided to double the final volume of each mix to 40uL instead of 20uL. Since we knew the concentration of each plasmid and also knew we needed 2500 ng, we cross multiplied to find the uL of DNA we would need for each.

Concentration Calculations:
310.4 ng/1 uL = 2500ng/x uL
x = 8.05 uL

Here is a chart indicating how much of each we used for the 2 mixes.

[[Image: DoubledigestRFPTetR.png]]

On the Promega website, we found a list of compatible buffers for double digestions. Buffers E and H are compatible for the Tet plasmid and RFP plasmid digestions respectively. We fortunately have both buffers in stock.

[[Image:BamEcoR1.png]] [[Image:NcoEcoR1.png]]

We waited for our waited for the restriction enzymes to arrive and in the meantime, we transformed the plasmids with pLab+RBS, pBAd, and pBad+RBS into ''E.coli'' cells. We want the ''E.coli'' cells to replicate the plasmids with these genes because we have a limited amount of genes. [http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_Transformation.html Zippy Plasmid Transformation]

Once our oligos and restriction enzymes arrived, we got to work right away!

'''PCR (Polymerase Chain Reaction)'''

We calculated how much plasmid we will need for the PCR. We decided to dilute the concentration of our plasmids a hundred fold so that we could get pipetable amounts.

''Change in Concentrations''
310.4 ng/uL x 1/100 (dilution factor) = 3.104 ng/uL (RFP)
163.6 ng/uL x 1/100 (dilution factor) = 0.164 ng/uL (Tet)

''Example shown with RFP''
3.104 ng/1 uL = 1ng/ x uL, x = .32
For Tet, x = .61

We also calculated how much of each primer we would need with the following equation:

C1V1 =C2V2
(100 uM) (x) = (1 uM)( 100 uL)
x= 1 uL

[[Image: screen-capture-8.png]]

Each of the 5 of us made had 2 tubes (one for each reporter protein) because there were 5 different forward primers.To each tube, we added the template DNA (either RFP or Tet), water, the respective reverse primer (either RFP or Tet), and the forward primer depending on which 5mer we had chosen. My 5mer was CCAUC. Finally, we added the Master Mix last because this contained the enzyme (DNA polymerase) that would amplify our desired sequence.

[[Image: Pcrtubes.png]]

We followed the temperature cycles so that PCR could occur. The annealing temperature needed to be 5C less than the smallest melting temperature of the primers. Since the reverse primer of RFP had a melting temp of 66C, we set the temperature of the 3rd cycle on 61C.

[[Image: Temperaturecycle.png]]

'''tRNA Oligo Assembly'''

We needed to calculate the concentration of each of the oligo before carrying out the assembly. The stock concentration of each of the oligo was 100 uM. Since we wanted to have 5uM of the oligo in the test tube, and we know that final volume of the test tube is 20 uL, we just cross multiplied to find the amount we needed to add to the tubes to get this concentration.

(100uM)x = (5uM)(20uL)
x = 1 uL

We added 1 uL to the tubes.

IGEM Notebook

2009-06-11T21:31:56Z

ShPunjabi: /* Thursday, June 11, 2009 */

==Wednesday, June 3, 2009==

Romina Clemente and I are trying to find suitable reporter proteins to use. Yesterday, Leland Taylor and Alyndria Thompson were working on ways to insert the gene sequences into the plasmid. Upon seeing how they wanted to manipulated the reporter gene to include the logical clauses, we came up with a few criteria for the reporter genes we would use. The following criteria for genes are listed in order of the broadest aspect to look at to the narrowest aspect:
a) Doesn't contain restriction sites for the 4 restriction enzymes (EcoR1, Xbal, Spel, Pst1) used to cleave the Biobrick part out of the plasmid.
b) Contains 6 cutter restriction sites.
c) These restriction enzymes aren't blunt (cleave straight down at one spot).
d) These restriction sites are close to thge 5' (beginning) end of the sequence.
e) These enzymes are easiest to work with and cheapest.
We are finding the part numbers of the reporter genes we want to use (antibiotic resistance, fluorescence, LacZ) through our own GCAT because we know these ones work. We are then locating these parts on the parts registry [1] website. We copied and pasted the gene sequences we obtained from the registry onto the ApE software [2]. From here, we were able to generate a genetic map of each gene that outlined each restriction site that fit our criteria. We put each genetic map, alongside the part number used, into a Word document.

http://gcat.davidson.edu/GcatWiki/images/0/0e/Restriction_Site_Mapping_on_Reporter_Genes.doc

Later on in the day, we decided that the best way to test for suppression would involve placing the 5mers at the beginning of the reporter gene instead of inserting them into the reporter gene. To do this, we decided that the gene along with its start codon need to be expressed after the 5mer. We will insert a BioBrick plasmid with a 5mer and start codon that will be incorporated before the gene:
RBS-6-8nt-ATG-5mer--ATG-gene

Leland Taylor assembled the oligos that we will need for the BioBrick pieces.

In order to test whether the reporter protein would be expressed regardless of the 5mer being suppressed or not, we would need to remove the suppressor tRNAs from the cell.

Meanwhile, I helped figure out the coding sequence that we will need for the suppressor tRNAs that will bind to the 5mers. We decided that we would be using the following suppressor tRNAs on the Davidson side: CUAGU, CCCUC, CGGUC, CCAUC, and CCACU. We searched through several papers, with little result and finally emailed Dr. Christopher Anderson of UC Berkeley to request the DNA sequence. He emailed us back with a generic DNA code that we could use to create the tRNA; however, for each suppressor tRNA we would use, we would need to change base pairs in the anticodon loop.

The length of the tRNA is 92 nt. The anticodon loop is comprised of 9 nt. Once we have BioBricked the DNA sequence with 4 restriction sites and supplemental nucleotides, the length of the entire gene is 144 nucleotides. This is made up of the 92 nt +22 nt on each side of the sequence (44) + 4 nt on each side of the restriction ends (8).

At the end of the day, we decided to use tetracycline resistance and RFP as our reporter proteins for this tester experiment.

==Thursday, June 4, 2009==

This morning, I began the day by learning how to use the PEARL prgram. Olivia Ho-Shing sat with me and walked me through the program script. And then, Leland Taylor and Shashank Suresh had a problem they wanted us to solve. Leland explained that we need to make sure there is a stop codon in the gene if we fail to suppress the 5mer. The stop codon needs to be before we find another 5mer. They also wanted us to run through both the RFP and Tet Resistance genes to make sure that they did not contain any of the anticodon sequences from our 5 frameshift suppressor tRNAs so as to throw the translation out of frame. They further wanted us to confirm that if we do have successful suppression, there are no stop codons in the middle of the RFP and Tet Resistance genes. PEARL did not find any matches for the suppressor tRNAs within genes. We found 3 matches for stop codons UAA. Two of these occured at the end of the Tet and one at the end of RFP, where they should normally be to end translation into a protein.

Following this, Olivia Ho-Shing and I tried to determine how we can add BioBrick ends to our single stranded DNA sequences that code for the suppressor tRNAs. Our ultimate goal is to find complements for these single strands so they can be put into plasmids. We found the standard prefixes and suffixes for sequences that do not contain ATG (becuase this sequence is a functional RNA and will not be translated) and we placed these before and after the altered 92bp sequence that Dr. Anderson provided us with. The prefixes and suffixes are necessary because they are the extra nucleotides that will be mimicking a "restriction enzyme cutting site". The single strand appeared as below after the Bio Brick prefixes and suffixes were added:

22ntPREFIX--32nt--VARIABLE ANTICODON LOOP--51nt--21ntSUFFIX

We used the suppressor codon CUAGU as our example and placed its appropriate anticodon loop in the sequence. We put this into the lancelator that would give us the other strand as well as the oligos we would need for ideal construction of this double stranded DNA fragment to put into the plasmid. We recieved a total of 4 oligos, 2 of which were variable and 2 of which were constant for all 6 different suppressor tRNAs. Using this double strand, we simulated a restriction enzyme digestion by EcoRI and PstI. We took off the G at both ends of the top strand and then removed the first five nucleotides on each end of the bottom strand. This made the top strand longer than the bottom one. We then proceeded to take the two variable strands and changed the anticodon loop nt for each suppressor tRNA we used.

Around lunchtime, we encountered a problem with the BioBrick scar in the "ATG-5mer subpiece" of the test project. It turns out that after annealing the sticky ends of the Xba1 and Spe1 sites, the scar created had a stop codon TAG (UAG) that was in frame with the suppressed codon. When the Xba1 site on insert anneals to the Spe1 site on the plasmid (the green portions), we get a scar that reads TACTAG.
[[Image:screen-capture.png]]

For the afternoon, Olivia Ho-Shing and I finished assembly of the oligos of the suppressor tRNA codes. We assembled the document below:

http://gcat.davidson.edu/GcatWiki/images/5/5e/TRNAoligostoOrder.doc

We were assigned tasks of creating "cartoons" to illustrate the phenomenon that had occurred. No one could successfully do this however, because we could not understand how the piece had formed. The parts registry website provided wrong information on the restriction site of Spe1 which slowed our efficiency. This project turned into an effort to try to find two other different restriction enzymes that have complementary sticky ends other than Xba1 and Spe1. We want to get rid oc the scar that contains TACTAG.

We ended up deciding that a hybrid of two ideas was needed: restriction enzymes would need to be used to make several reporter gene sequences with different beginnings dependending on the suppressor tRNA sequence needed.

==Friday, June 5, 2009==

Our goal for this morning was to discuss the hybrid PCR approach to assemble the part that comes before the gene we wanted to express. We decided that we would have a BioBrick prefix and a 5mer followed by a sequence of nucleotides whose identity was up to us. Using PCR we would get the second double strand if we have the following:

BioBrick Prefix--ATG--5mer--gene until we get to restriction site

The PCR primer would attach to the ATG and the 5mer and would replicate the complementary base pairs to make a double stranded DNA sequence. As several of us were confused about PCR methods, we looked at the online link provided by Alyndria Thompson's link to the PCR interactive website which was very helpful. We also did some research to discover that the restriction enzyme BseRI (which is contained in the YFP that we wanted to alter) is not compatible with any other restriction enzyme. This is good because we did not want to plasmid containing this gene to close up with Xba1 once we digested it.

The team experienced some confusion with the hybrid idea and spoke to Clif Davis of Missouri Western University on the phone. He told us that there will be one part that is a fragment of the beginning of some nucleotides as a restriction cleaving site, ATG, the logical clause, and the start of the YFP gene that will be placed into a plasmid containing a space. To the left of the space will be the BioBrick Prefix and the right of the space will contain the rest of the genes in the YFP. Therefore, we decided not to alter any BioBrick ends and just look at restriction sites at the beginning of reporter proteins. The small fragment that will be placed into the plasmid will be made by PCR. The PCR will attach a primer onto the ATG and 5mer and replicate.

While we were discussing this, we expanded upon the idea that we can do whatever we want with the "suffix" portion between the 5mer and the beginning of YFP. We decided it would be best to remove ATG and add the first 5 amino acids and the restriction enzyme site. Along with this, if we have the 5mer coding for a certain amino acid (ie methionine) we could replace another codon that codes for methionine WITH that 5mer so long as this codon is contained within the first 15 nt. This way, we would not be altering the protein.

Olivia Ho-Shing came up with an idea of cascading PCR effect for longer logical clauses. Post translational modification of the protein was another one of her ideas. This applies because although we may code for the protein in a given way, the protein may modify or fix itself up to perform a slightly different function in its tertiary form.

Dr. Campbell suggested that we can look up restriction enzymes 400-500 base pairs into the reporter protein we would like to use; however, we have more flexibility in our procedure if we choose more "hardy" cutters. So instead, we could cut with EcoRI instead of with Xba1.

During the afternoon, we set about three tasks:
1) Which primers to use
2) Which promoters to use
3) Which restriction enzymes and how they will determine use of reporter proteins

Olivia Ho-Shing and I began looking up which restriction enzymes were strong so we could base our reporters on that. We began with Tet and RFP since those were the two reporters we had started out with choosing. I found a website that provided enzyme cleavage efficiency based upon the proximity of the site to the end of the PCR fragment that contains it. We discovered the following based upon the attached documents:

http://gcat.davidson.edu/GcatWiki/images/8/8f/Restriction_Enzyme_Cleavage_Efficiency.doc
http://gcat.davidson.edu/GcatWiki/images/9/9a/07Jun09_RE_Cutting_Efficiency_oh.doc

We decided that these two reporters contained efficient enzymes in a good quantity to choose from. On Monday, we will look through these enzymes once more and finalize which enzymes we would like to use with RFP and Tet.

==Sunday, June 7, 2009==

We did preparations for the mini prep this evening. The following notes are courtesy of Olivia Ho-Shing.

1. Sterically transfer ~2mL LB+AMP (AMP are ampicillin resistant plasmids) *Never put fluid back into bottle once it has entered pipette*
2. Use forceps to grab toothpick, scrap some frozen cells, rub in tube with top

'''Tubes'''

S03511 (2)

K091111

B0030

I715039

K091112

I715039

J31007 (2)

B0034

E1010

S03710

==Monday, June 8, 2009==

We reviewed the PCR process this morning as well as the restriction enzyme cleavage process.

Then, Leland Taylor, Olivia Ho-Shing and I all worked on converting the tRNA oligos we made to the template that Missouri Western used. The oligos we had previously made were different from the ones that Dr. Anderson had originally emailed us because further communication between Dr. Anderson and Missouri Western resulted in a better template.

We tried several times to minimize the number of oligos while keeping their individual lengths under 80bp. We managed to do this in the end by creating 7 oligos, 2 of which are variable between the suppressor tRNAs.
We reviewed the following document and finalized the oligos that we needed to order for the frameshift suppressor tRNAs. We decided to keep both the 10bp and 9bp anticodons of CCAUC.

I also looked up the amino acids that corresponded to the FS (frameshift suppressor) tRNAs for future use:

1. CUAGU = Leu
2. CCCUC = Pro
3. CGGUC = Arg
4. CCACU = Pro
5. CCAUC* = Pro
*9 and 10 bp anticodons

Also, we have decided to use NcoI as the restriction enzyme for RFP. This cleavage site occurs 419 bp into the gene. We have decided to use BamHI as the enzyme for Tetracycline Resistance and the cleavage site occurs 290bp into the gene. Both of these enzymes are very powerful and hardy.

We also looked over Missouri Western's tRNA oligos and primer oligos which we approved.
Our oligos were also approved by Missouri Western and Dr. Campbell ordered them while we supervised.

In the afternoon we did the miniprep. The mini prep directions were as appear in the link below.

http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_MiniPrep.html

We then worked with the Nano Prep to dilute our solution of DNA to an absorption of 1.5 and then we made labels for the tubes. The chart below shows relevant information we obtained from the NanoDrop for each of the reporter plasmids.

[[Image: Screen-capture-6.png]]

==Tuesday, June 9, 2009==

'''Preparations for restriction enzymes digestion of plasmids'''

Purpose: To determine the size of the plasmid inserts and ensure that plasmids are what we extracted from the cells during the mini-prep.

Things we need:
DNA ~3uL
Buffer 10x (Want to dilute to 1x) so add 2uL
Enzyme 1 ~1uL
Enzme 2 ~1uL
Water ~13uL
Final Volunter about 20 uL.

Need to choose the right buffer for the restriction enzymes. All buffers come in 10x (10 times more concentrated than what you want it to be when you use it). [http://gcat.davidson.edu/GcatWiki/index.php/Davidson_Missouri_W/Double_Digest_Guide Buffer Website]

Total volume of enzymes has to be 10% or less than final volume (most volume 2uL). Barely submerge the pipet tip into the solution when pipeting the enzymes.
Star (*) activity: Restriction enzyme stops cutting specifically and starts cutting everywhere.

DNA can't be seen by itself so we add EtBr (the bigger the insert, the more will bind) so bigger strands appear brighter. samples.

The order of entry doesn't matter, but make sure to enter the enzymes last.

Will have lots of labeled tubes and will put 3uL of appropriate plasmids into the tubes.
In the meantime, we'll make a "Master Mix" of all the other ingredients in a bigger tube. Always make enough volume for one extra tube.

Enzymes usually have an optimal temperature of 37C. We will let this reaction go for an hour and then we will run it on a gel.

'''Gel Electrophoresis Protocol'''

Website online for ideal gel concentration for resolving different sizes of molecules [http://gcat.davidson.edu/iGEM08/gelwebsite/gelwebsite.html Gel Concentration Website].
See protocol on the Davidson/Missouri Western Wookie for running molecules in the gel and how to digest with restriction enzymes.

'''What's Next?'''

1. 5' end additions
a. Using PCR
b. Cut at the restriction sites EcoR1 and either Bam H1 or Nco1
c. Cut plasmid and reporter with same restriction enzymes as above
d. Run a gel and purify "keeper" DNA (Ligate transform, screen, sequence)
e. Then, we take the "keeper" DNA and combine it with the insert from part

We can have steps d and e ready in advance.

2. tRNAs
a. Assemble the oligos into genes (Requires lots of calculations beforehand, but the actual steps )
b. Plasmids cut up with EcoR1 and Pst1 '''WE CAN USE THE "WASTE" PLASMID FROM THE RESTRICTION ENZYME DIGEST'''
c. Gel purify the plasmids (Ligate transform, screen, sequence)

The following tables include Master Mix measurements, agarose gel concentrations and buffers.
http://gcat.davidson.edu/GcatWiki/images/0/08/Shamitapic3.png
http://gcat.davidson.edu/GcatWiki/images/d/d1/Shamitapic4.png
http://gcat.davidson.edu/GcatWiki/images/6/6f/Shamitapic5.png

We made the Master Mix with our DNA and put it in the incubator at 10:40 AM.

'''Preparing the Gels'''

Now, we will be making 2 gels, 1 at 0.8% and 2.5% (weight to volume percent). The gel volume is 60mL.

'''Definition of 1% solution:
1 gram of agarose/ 100mL buffer'''

So for a 0.8% solution: 0.8 grams agarose/100mL buffer AND we need 60mL (fixed volume for casts run) so multiply that by 0.6.
Final concentration:

.48 grams agarose/60mL buffer

1 uL of EtBr stock for 60mL of gel.
EtBr is in the buffer so it is constantly replenishing itself even though it is moving toward one end in order to make bands on the other end visible.

After Dr. Campbell's demonstration, we each helped prepare a 0.8% buffer solution using the agarose powder, buffer and EtBr. We mixed together the first two substances in a 200mL Erlenmeyer flask and heated it for 120 seconds in the microwave. We then added EtBr and poured the gel solution into the gel cast.

'''Agar Plate'''

After lunch, we grew some more cells for each of the 11 parts in an agar plate labeled "LBAMP-AMC-9 June" so that so would have extra cells that could be stored for up to 2 weeks.
We made small circles on the agar plate and numbered them 1-11. The numbers corresponded to the following parts. We put the plate in the incubator overnight to let the cells grow.

1. B0030 AMC
2. K091111
3. S03710
4. I715039 OEH
5. S03511 ORH
6. I715039 AST
7. B0030 SDP
8. K091112
9. J31007 LJH
10. J31007 REC
11. S03511

'''Unloading the Gels'''

The following shows which parts we entered in which gels:

[[Image: screen-capture-2.png]]

We unloaded the 2.5% agarose first and after observing the bands under a UV light, put the gel into the BioRad machine to observe the bands. The image below is of a Molecular Weight Marker that shows the number of bp in control fragments of DNA according to the distance they travel down the gel.

[[Image: screen-capture-1.png]]

We obtained the following printout for the first gel that was 2.5% Agarose.
[[Image: 2009-06-09_14hr_44min.jpg]]

The bands in the first well at the bottom are those of the molecular weight marker. We skipped the lane above (to the right) of the MWM and entered the rest of the inserts as described in the above table. A light band is visible for each of the 4 other wells. In Lanes 3 and 4 we concluded we are supposed to have approximately 45 bp because the insert is 15bp + 30 bp on either side of the insert (Xba1 and Spe1 restrictions sites + sticky ends + extra bases). We used the same logic for Lanes 5 and 6 which are supposed to contain approximately 86 base pairs.

One discrepancy in this gel, however, is that Lanes 5 and 6, which according to their labels respectively contain the LacIQ promoter and the Lac IQ1 promoter. According to the information online at the parts registry website, pLacIQ1 should be the same length as pLacIQ. The gels do not show this, but instead show that the sample in Lane 5 is smaller than that in Lane 6 because Lane 5 traveled further. Leland Taylor and Romina Clemente sent out an email to Pallavi Penumetcha to ask about this because she had written a paper about this.

We then unloaded the 0.8% agarose gel and then placed this in the BioRad machine under the same protocol as above. The following image is the 0.8% Agarose.

[[Image:2009-06-09_15hr_21min.jpg]]

We observed that the bands in Lane 1 (which is now at the top) are examples of high intensity bands. The plasmids contained in the original well are twice the intensity of the insert.

In Lane 5, however, we observed several different bands. This could be due to the extra DNA that was sitting on the top of the well. We decided to re-expose the gel to get better observations on Lanes 3 and 4 because the bands were not as intense. We anticipated seeing a band at about 250 bp because this was the size of the insert (plus extra bp). After re-exposing the gel, we did not find any difference and still did not see any bands in these lanes. We have decided to redo the gel on the part S03511 and so we prepared the master mix with that DNA and incubated it so that we could run the gel tomorrow.

We also cut out the "empty" plasmids from the gel so that we could use this as vectors for the coding sequence of the suppressor tRNAs.

Tomorrow we will be planning out specific tasks for people once the oligos arrive.

==Wednesday, June 10, 2009==

We began this morning by making the agarose gel with the same protocol as yesterday. This time, since we are just re-doing both S03511 inserts, we will have a 1.8% agarose gel.

'''OUTLINE: WHERE DO WE STAND?'''

Yesterday
a. Isolated empty plasmids
b. Verified most inserts
c. Waiting for:
>tRNA oligos
>Primers
a. Will amplify the reporter gene in the plasmid itself

Next?
a. When Primers Arrive
> Amplify the reporter genes with primers (PCR takes ~3 hours)
> Calculate how much reporter plasmid we will need (Start with 50x more than we actually will need)
> Cut amplified sequence (EcoR1 and BamH1/Nco1) then purify what we need
> Cut reporter plasmid (EcoR1 and BamH1/Nco1) then purify what we need
> Ligate amplified sequence and reporter plasmid
b. When tRNA oligos arrive
> Calculate how much of each oligo we will need (takes a while...)
> "Boil and cool" tRNA oligos together
> Ligate fragments (dsDNA) into plasmids we isolated yesterday (~5 minutes)
> Transform the plasmids (~20 minutes)

* LOOK THROUGH LIGATION PROTOCOL (How to assemble genes)
* LOOK THROUGH TRANSFORMATION PROTOCOL

''What we need to get done today'':
1. Prepare the control for the Tet Resistance by BioBricking the part pLac+RBS (S03511) with the TetA (J31007).
2. Check the part number for RFP and also for pLac+RBS+RFP
3. Read through ligation protocol
4. See how many nanograms of plasmid we will need for the tRNAs and for the inserts and multiply this number by 50.
> Check if we have enough plasmids from our mini-prep
5. Purify the plasmids from the gel

After we made this outline, Leland, Olivia and I analyzed the gel we ran this morning and did not see a more than one band in the 2 lanes that contained DNA from S03511 (image below). Since we did not see multiple bands in yesterday's gel either, we conclude that there is something wrong with the DNA rather than with the restriction enzymes that we used to cut it. We concluded that there is probably not an insert in this plasmid both the part and the plasmid are useless.

[[Image: 2009-06-10 10hr 30min.jpg]]

Now, we do not have an immediately accessible promoter. We have decided to order the pBAD promoter from Missouri Western and we will attach the RBS+Tet to this plasmid. Our insert for this will be the larger gene because it is easier to manipulate in the gel than the small gene.

Alyndria Thompson and Romina Clemente are figuring out the size of the plasmids including the reporter genes.

After lunch we performed several steps to isolate and purify the gel to get the empty plasmids will be the vectors for the tRNA DNA. Then, we used the NanoDrop to assess the purity of our DNA. We found that the plasmids were more pure than we had expected. The following values were obtained:

Abs 1.850
A-260 10 mm path 0.172
A-280 10 mm path 0.075
260/280 2.28
260/230 0.09

We then followed the protocol for preparing the agar plates. We put the plate with the agar solution in the Autoclave.

While the solution was in the autoclave, we discussed that we don't really need a promoter in front of RBS+Tet for the gene to be expressed because Tet expression is so strong that the gene would translate without a promoter. However, it would be a good idea to have the promoter because if it was possible to reinforce the translation of the reporter protein, we wanted to do that.

We found that there were some tubes in the lab fridge that contained the pBAD and pLac promoters with RBS. We decided to use these as the promoter and RBS for our experiments. The pLac promoter will be used for RFP and the pBad promoter used with Tet Resistance. We decided to use the pBad with Tet because it is weaker in comparison to pLac. As expression of the Tet gene can be toxic to a cell, we do not want to overexpress the gene with a stronger promoter.

Once the agar solution was sterilized in the Autoclave, we removed it, cooled it, and pipetted the antibiotic resistance into it. We then poured the solution into the plates, removed any bubbles, and let the gel form.

Alyndria Thompson and Romina Clemente concluded that we had more than enough recieving vectors for both the RFP and Tetracycline genes. Since we need 50 nanograms, it is wise to have at least 50 times that much that we can work with, so we need at least 2,500 ng.

RFP 1: 77.6 x 4 = 310.4 ng/uL
x 20 uL = 3,272 ng
RFP 2: 59.5 x 4 = 238 ng/uL
x 20 uL = 4,760 ng
Tet: 40.9 x 4 = 163.6 ng/uL
x 20 uL = 3,272 ng

According to these calculations we have more than 50 times what we need for both instances of RFP and Tet.

Tomorrow, we can prepare the Master Mix for the digestions of both reporter plamids and then add the restriction enzymes once they arrive. Then, we can follow through with the rest of the plans we had made above in the section titles "Next?".

==Thursday, June 11, 2009==

We began this morning by calculating how many uL we would need for each of the mixes for digesting our reporter plasmids. We decided to double the final volume of each mix to 40uL instead of 20uL. Since we knew the concentration of each plasmid and also knew we needed 2500 ng, we cross multiplied to find the uL of DNA we would need for each.

Concentration Calculations:
310.4 ng/1 uL = 2500ng/x uL
x = 8.05 uL

Here is a chart indicating how much of each we used for the 2 mixes.

[[Image: DoubledigestRFPTetR.png]]

On the Promega website, we found a list of compatible buffers for double digestions. Buffers E and H are compatible for the Tet plasmid and RFP plasmid digestions respectively. We fortunately have both buffers in stock.

[[Image:BamEcoR1.png]] [[Image:NcoEcoR1.png]]

We waited for our waited for the restriction enzymes to arrive and in the meantime, we transformed the plasmids with pLab+RBS, pBAd, and pBad+RBS into ''E.coli'' cells. We want the ''E.coli'' cells to replicate the plasmids with these genes because we have a limited amount of genes. [http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_Transformation.html Zippy Plasmid Transformation]

Once our oligos and restriction enzymes arrived, we got to work right away!

'''PCR (Polymerase Chain Reaction)'''

We calculated how much plasmid we will need for the PCR. We decided to dilute the concentration of our plasmids a hundred fold so that we could get pipetable amounts.

''Change in Concentrations''
310.4 ng/uL x 1/100 (dilution factor) = 3.104 ng/uL (RFP)
163.6 ng/uL x 1/100 (dilution factor) = 0.164 ng/uL (Tet)

''Example shown with RFP''
3.104 ng/1 uL = 1ng/ x uL, x = .32
For Tet, x = .61

We also calculated how much of each primer we would need with the following equation:

C1V1 =C2V2
(100 uM) (x) = (1 uM)( 100 uL)
x= 1 uL

[[Image: screen-capture-8.png]]

Each of the 5 of us made had 2 tubes (one for each reporter protein) because there were 5 different forward primers.To each tube, we added the template DNA (either RFP or Tet), water, the respective reverse primer (either RFP or Tet), and the forward primer depending on which 5mer we had chosen. My 5mer was CCAUC. Finally, we added the Master Mix last because this contained the enzyme (DNA polymerase) that would amplify our desired sequence.

[[Image: Pcrtubes.png]]

We followed the temperature cycles so that PCR could occur. The annealing temperature needed to be 5C less than the smallest melting temperature of the primers. Since the reverse primer of RFP had a melting temp of 66C, we set the temperature of the 3rd cycle on 61C.

[[Image: Temperaturecycle.png]]

'''tRNA Oligo Assembly'''

We needed to calculate the concentration of each of the oligo before carrying out the assembly. The stock concentration of each of the oligo was 100 uM. Since we wanted to have 5uM of the oligo in the test tube, and we know that final volume of the test tube is 20 uL.

IGEM Notebook

2009-06-11T21:14:21Z

ShPunjabi: /* Thursday, June 11, 2009 */

==Wednesday, June 3, 2009==

Romina Clemente and I are trying to find suitable reporter proteins to use. Yesterday, Leland Taylor and Alyndria Thompson were working on ways to insert the gene sequences into the plasmid. Upon seeing how they wanted to manipulated the reporter gene to include the logical clauses, we came up with a few criteria for the reporter genes we would use. The following criteria for genes are listed in order of the broadest aspect to look at to the narrowest aspect:
a) Doesn't contain restriction sites for the 4 restriction enzymes (EcoR1, Xbal, Spel, Pst1) used to cleave the Biobrick part out of the plasmid.
b) Contains 6 cutter restriction sites.
c) These restriction enzymes aren't blunt (cleave straight down at one spot).
d) These restriction sites are close to thge 5' (beginning) end of the sequence.
e) These enzymes are easiest to work with and cheapest.
We are finding the part numbers of the reporter genes we want to use (antibiotic resistance, fluorescence, LacZ) through our own GCAT because we know these ones work. We are then locating these parts on the parts registry [1] website. We copied and pasted the gene sequences we obtained from the registry onto the ApE software [2]. From here, we were able to generate a genetic map of each gene that outlined each restriction site that fit our criteria. We put each genetic map, alongside the part number used, into a Word document.

http://gcat.davidson.edu/GcatWiki/images/0/0e/Restriction_Site_Mapping_on_Reporter_Genes.doc

Later on in the day, we decided that the best way to test for suppression would involve placing the 5mers at the beginning of the reporter gene instead of inserting them into the reporter gene. To do this, we decided that the gene along with its start codon need to be expressed after the 5mer. We will insert a BioBrick plasmid with a 5mer and start codon that will be incorporated before the gene:
RBS-6-8nt-ATG-5mer--ATG-gene

Leland Taylor assembled the oligos that we will need for the BioBrick pieces.

In order to test whether the reporter protein would be expressed regardless of the 5mer being suppressed or not, we would need to remove the suppressor tRNAs from the cell.

Meanwhile, I helped figure out the coding sequence that we will need for the suppressor tRNAs that will bind to the 5mers. We decided that we would be using the following suppressor tRNAs on the Davidson side: CUAGU, CCCUC, CGGUC, CCAUC, and CCACU. We searched through several papers, with little result and finally emailed Dr. Christopher Anderson of UC Berkeley to request the DNA sequence. He emailed us back with a generic DNA code that we could use to create the tRNA; however, for each suppressor tRNA we would use, we would need to change base pairs in the anticodon loop.

The length of the tRNA is 92 nt. The anticodon loop is comprised of 9 nt. Once we have BioBricked the DNA sequence with 4 restriction sites and supplemental nucleotides, the length of the entire gene is 144 nucleotides. This is made up of the 92 nt +22 nt on each side of the sequence (44) + 4 nt on each side of the restriction ends (8).

At the end of the day, we decided to use tetracycline resistance and RFP as our reporter proteins for this tester experiment.

==Thursday, June 4, 2009==

This morning, I began the day by learning how to use the PEARL prgram. Olivia Ho-Shing sat with me and walked me through the program script. And then, Leland Taylor and Shashank Suresh had a problem they wanted us to solve. Leland explained that we need to make sure there is a stop codon in the gene if we fail to suppress the 5mer. The stop codon needs to be before we find another 5mer. They also wanted us to run through both the RFP and Tet Resistance genes to make sure that they did not contain any of the anticodon sequences from our 5 frameshift suppressor tRNAs so as to throw the translation out of frame. They further wanted us to confirm that if we do have successful suppression, there are no stop codons in the middle of the RFP and Tet Resistance genes. PEARL did not find any matches for the suppressor tRNAs within genes. We found 3 matches for stop codons UAA. Two of these occured at the end of the Tet and one at the end of RFP, where they should normally be to end translation into a protein.

Following this, Olivia Ho-Shing and I tried to determine how we can add BioBrick ends to our single stranded DNA sequences that code for the suppressor tRNAs. Our ultimate goal is to find complements for these single strands so they can be put into plasmids. We found the standard prefixes and suffixes for sequences that do not contain ATG (becuase this sequence is a functional RNA and will not be translated) and we placed these before and after the altered 92bp sequence that Dr. Anderson provided us with. The prefixes and suffixes are necessary because they are the extra nucleotides that will be mimicking a "restriction enzyme cutting site". The single strand appeared as below after the Bio Brick prefixes and suffixes were added:

22ntPREFIX--32nt--VARIABLE ANTICODON LOOP--51nt--21ntSUFFIX

We used the suppressor codon CUAGU as our example and placed its appropriate anticodon loop in the sequence. We put this into the lancelator that would give us the other strand as well as the oligos we would need for ideal construction of this double stranded DNA fragment to put into the plasmid. We recieved a total of 4 oligos, 2 of which were variable and 2 of which were constant for all 6 different suppressor tRNAs. Using this double strand, we simulated a restriction enzyme digestion by EcoRI and PstI. We took off the G at both ends of the top strand and then removed the first five nucleotides on each end of the bottom strand. This made the top strand longer than the bottom one. We then proceeded to take the two variable strands and changed the anticodon loop nt for each suppressor tRNA we used.

Around lunchtime, we encountered a problem with the BioBrick scar in the "ATG-5mer subpiece" of the test project. It turns out that after annealing the sticky ends of the Xba1 and Spe1 sites, the scar created had a stop codon TAG (UAG) that was in frame with the suppressed codon. When the Xba1 site on insert anneals to the Spe1 site on the plasmid (the green portions), we get a scar that reads TACTAG.
[[Image:screen-capture.png]]

For the afternoon, Olivia Ho-Shing and I finished assembly of the oligos of the suppressor tRNA codes. We assembled the document below:

http://gcat.davidson.edu/GcatWiki/images/5/5e/TRNAoligostoOrder.doc

We were assigned tasks of creating "cartoons" to illustrate the phenomenon that had occurred. No one could successfully do this however, because we could not understand how the piece had formed. The parts registry website provided wrong information on the restriction site of Spe1 which slowed our efficiency. This project turned into an effort to try to find two other different restriction enzymes that have complementary sticky ends other than Xba1 and Spe1. We want to get rid oc the scar that contains TACTAG.

We ended up deciding that a hybrid of two ideas was needed: restriction enzymes would need to be used to make several reporter gene sequences with different beginnings dependending on the suppressor tRNA sequence needed.

==Friday, June 5, 2009==

Our goal for this morning was to discuss the hybrid PCR approach to assemble the part that comes before the gene we wanted to express. We decided that we would have a BioBrick prefix and a 5mer followed by a sequence of nucleotides whose identity was up to us. Using PCR we would get the second double strand if we have the following:

BioBrick Prefix--ATG--5mer--gene until we get to restriction site

The PCR primer would attach to the ATG and the 5mer and would replicate the complementary base pairs to make a double stranded DNA sequence. As several of us were confused about PCR methods, we looked at the online link provided by Alyndria Thompson's link to the PCR interactive website which was very helpful. We also did some research to discover that the restriction enzyme BseRI (which is contained in the YFP that we wanted to alter) is not compatible with any other restriction enzyme. This is good because we did not want to plasmid containing this gene to close up with Xba1 once we digested it.

The team experienced some confusion with the hybrid idea and spoke to Clif Davis of Missouri Western University on the phone. He told us that there will be one part that is a fragment of the beginning of some nucleotides as a restriction cleaving site, ATG, the logical clause, and the start of the YFP gene that will be placed into a plasmid containing a space. To the left of the space will be the BioBrick Prefix and the right of the space will contain the rest of the genes in the YFP. Therefore, we decided not to alter any BioBrick ends and just look at restriction sites at the beginning of reporter proteins. The small fragment that will be placed into the plasmid will be made by PCR. The PCR will attach a primer onto the ATG and 5mer and replicate.

While we were discussing this, we expanded upon the idea that we can do whatever we want with the "suffix" portion between the 5mer and the beginning of YFP. We decided it would be best to remove ATG and add the first 5 amino acids and the restriction enzyme site. Along with this, if we have the 5mer coding for a certain amino acid (ie methionine) we could replace another codon that codes for methionine WITH that 5mer so long as this codon is contained within the first 15 nt. This way, we would not be altering the protein.

Olivia Ho-Shing came up with an idea of cascading PCR effect for longer logical clauses. Post translational modification of the protein was another one of her ideas. This applies because although we may code for the protein in a given way, the protein may modify or fix itself up to perform a slightly different function in its tertiary form.

Dr. Campbell suggested that we can look up restriction enzymes 400-500 base pairs into the reporter protein we would like to use; however, we have more flexibility in our procedure if we choose more "hardy" cutters. So instead, we could cut with EcoRI instead of with Xba1.

During the afternoon, we set about three tasks:
1) Which primers to use
2) Which promoters to use
3) Which restriction enzymes and how they will determine use of reporter proteins

Olivia Ho-Shing and I began looking up which restriction enzymes were strong so we could base our reporters on that. We began with Tet and RFP since those were the two reporters we had started out with choosing. I found a website that provided enzyme cleavage efficiency based upon the proximity of the site to the end of the PCR fragment that contains it. We discovered the following based upon the attached documents:

http://gcat.davidson.edu/GcatWiki/images/8/8f/Restriction_Enzyme_Cleavage_Efficiency.doc
http://gcat.davidson.edu/GcatWiki/images/9/9a/07Jun09_RE_Cutting_Efficiency_oh.doc

We decided that these two reporters contained efficient enzymes in a good quantity to choose from. On Monday, we will look through these enzymes once more and finalize which enzymes we would like to use with RFP and Tet.

==Sunday, June 7, 2009==

We did preparations for the mini prep this evening. The following notes are courtesy of Olivia Ho-Shing.

1. Sterically transfer ~2mL LB+AMP (AMP are ampicillin resistant plasmids) *Never put fluid back into bottle once it has entered pipette*
2. Use forceps to grab toothpick, scrap some frozen cells, rub in tube with top

'''Tubes'''

S03511 (2)

K091111

B0030

I715039

K091112

I715039

J31007 (2)

B0034

E1010

S03710

==Monday, June 8, 2009==

We reviewed the PCR process this morning as well as the restriction enzyme cleavage process.

Then, Leland Taylor, Olivia Ho-Shing and I all worked on converting the tRNA oligos we made to the template that Missouri Western used. The oligos we had previously made were different from the ones that Dr. Anderson had originally emailed us because further communication between Dr. Anderson and Missouri Western resulted in a better template.

We tried several times to minimize the number of oligos while keeping their individual lengths under 80bp. We managed to do this in the end by creating 7 oligos, 2 of which are variable between the suppressor tRNAs.
We reviewed the following document and finalized the oligos that we needed to order for the frameshift suppressor tRNAs. We decided to keep both the 10bp and 9bp anticodons of CCAUC.

I also looked up the amino acids that corresponded to the FS (frameshift suppressor) tRNAs for future use:

1. CUAGU = Leu
2. CCCUC = Pro
3. CGGUC = Arg
4. CCACU = Pro
5. CCAUC* = Pro
*9 and 10 bp anticodons

Also, we have decided to use NcoI as the restriction enzyme for RFP. This cleavage site occurs 419 bp into the gene. We have decided to use BamHI as the enzyme for Tetracycline Resistance and the cleavage site occurs 290bp into the gene. Both of these enzymes are very powerful and hardy.

We also looked over Missouri Western's tRNA oligos and primer oligos which we approved.
Our oligos were also approved by Missouri Western and Dr. Campbell ordered them while we supervised.

In the afternoon we did the miniprep. The mini prep directions were as appear in the link below.

http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_MiniPrep.html

We then worked with the Nano Prep to dilute our solution of DNA to an absorption of 1.5 and then we made labels for the tubes. The chart below shows relevant information we obtained from the NanoDrop for each of the reporter plasmids.

[[Image: Screen-capture-6.png]]

==Tuesday, June 9, 2009==

'''Preparations for restriction enzymes digestion of plasmids'''

Purpose: To determine the size of the plasmid inserts and ensure that plasmids are what we extracted from the cells during the mini-prep.

Things we need:
DNA ~3uL
Buffer 10x (Want to dilute to 1x) so add 2uL
Enzyme 1 ~1uL
Enzme 2 ~1uL
Water ~13uL
Final Volunter about 20 uL.

Need to choose the right buffer for the restriction enzymes. All buffers come in 10x (10 times more concentrated than what you want it to be when you use it). [http://gcat.davidson.edu/GcatWiki/index.php/Davidson_Missouri_W/Double_Digest_Guide Buffer Website]

Total volume of enzymes has to be 10% or less than final volume (most volume 2uL). Barely submerge the pipet tip into the solution when pipeting the enzymes.
Star (*) activity: Restriction enzyme stops cutting specifically and starts cutting everywhere.

DNA can't be seen by itself so we add EtBr (the bigger the insert, the more will bind) so bigger strands appear brighter. samples.

The order of entry doesn't matter, but make sure to enter the enzymes last.

Will have lots of labeled tubes and will put 3uL of appropriate plasmids into the tubes.
In the meantime, we'll make a "Master Mix" of all the other ingredients in a bigger tube. Always make enough volume for one extra tube.

Enzymes usually have an optimal temperature of 37C. We will let this reaction go for an hour and then we will run it on a gel.

'''Gel Electrophoresis Protocol'''

Website online for ideal gel concentration for resolving different sizes of molecules [http://gcat.davidson.edu/iGEM08/gelwebsite/gelwebsite.html Gel Concentration Website].
See protocol on the Davidson/Missouri Western Wookie for running molecules in the gel and how to digest with restriction enzymes.

'''What's Next?'''

1. 5' end additions
a. Using PCR
b. Cut at the restriction sites EcoR1 and either Bam H1 or Nco1
c. Cut plasmid and reporter with same restriction enzymes as above
d. Run a gel and purify "keeper" DNA (Ligate transform, screen, sequence)
e. Then, we take the "keeper" DNA and combine it with the insert from part

We can have steps d and e ready in advance.

2. tRNAs
a. Assemble the oligos into genes (Requires lots of calculations beforehand, but the actual steps )
b. Plasmids cut up with EcoR1 and Pst1 '''WE CAN USE THE "WASTE" PLASMID FROM THE RESTRICTION ENZYME DIGEST'''
c. Gel purify the plasmids (Ligate transform, screen, sequence)

The following tables include Master Mix measurements, agarose gel concentrations and buffers.
http://gcat.davidson.edu/GcatWiki/images/0/08/Shamitapic3.png
http://gcat.davidson.edu/GcatWiki/images/d/d1/Shamitapic4.png
http://gcat.davidson.edu/GcatWiki/images/6/6f/Shamitapic5.png

We made the Master Mix with our DNA and put it in the incubator at 10:40 AM.

'''Preparing the Gels'''

Now, we will be making 2 gels, 1 at 0.8% and 2.5% (weight to volume percent). The gel volume is 60mL.

'''Definition of 1% solution:
1 gram of agarose/ 100mL buffer'''

So for a 0.8% solution: 0.8 grams agarose/100mL buffer AND we need 60mL (fixed volume for casts run) so multiply that by 0.6.
Final concentration:

.48 grams agarose/60mL buffer

1 uL of EtBr stock for 60mL of gel.
EtBr is in the buffer so it is constantly replenishing itself even though it is moving toward one end in order to make bands on the other end visible.

After Dr. Campbell's demonstration, we each helped prepare a 0.8% buffer solution using the agarose powder, buffer and EtBr. We mixed together the first two substances in a 200mL Erlenmeyer flask and heated it for 120 seconds in the microwave. We then added EtBr and poured the gel solution into the gel cast.

'''Agar Plate'''

After lunch, we grew some more cells for each of the 11 parts in an agar plate labeled "LBAMP-AMC-9 June" so that so would have extra cells that could be stored for up to 2 weeks.
We made small circles on the agar plate and numbered them 1-11. The numbers corresponded to the following parts. We put the plate in the incubator overnight to let the cells grow.

1. B0030 AMC
2. K091111
3. S03710
4. I715039 OEH
5. S03511 ORH
6. I715039 AST
7. B0030 SDP
8. K091112
9. J31007 LJH
10. J31007 REC
11. S03511

'''Unloading the Gels'''

The following shows which parts we entered in which gels:

[[Image: screen-capture-2.png]]

We unloaded the 2.5% agarose first and after observing the bands under a UV light, put the gel into the BioRad machine to observe the bands. The image below is of a Molecular Weight Marker that shows the number of bp in control fragments of DNA according to the distance they travel down the gel.

[[Image: screen-capture-1.png]]

We obtained the following printout for the first gel that was 2.5% Agarose.
[[Image: 2009-06-09_14hr_44min.jpg]]

The bands in the first well at the bottom are those of the molecular weight marker. We skipped the lane above (to the right) of the MWM and entered the rest of the inserts as described in the above table. A light band is visible for each of the 4 other wells. In Lanes 3 and 4 we concluded we are supposed to have approximately 45 bp because the insert is 15bp + 30 bp on either side of the insert (Xba1 and Spe1 restrictions sites + sticky ends + extra bases). We used the same logic for Lanes 5 and 6 which are supposed to contain approximately 86 base pairs.

One discrepancy in this gel, however, is that Lanes 5 and 6, which according to their labels respectively contain the LacIQ promoter and the Lac IQ1 promoter. According to the information online at the parts registry website, pLacIQ1 should be the same length as pLacIQ. The gels do not show this, but instead show that the sample in Lane 5 is smaller than that in Lane 6 because Lane 5 traveled further. Leland Taylor and Romina Clemente sent out an email to Pallavi Penumetcha to ask about this because she had written a paper about this.

We then unloaded the 0.8% agarose gel and then placed this in the BioRad machine under the same protocol as above. The following image is the 0.8% Agarose.

[[Image:2009-06-09_15hr_21min.jpg]]

We observed that the bands in Lane 1 (which is now at the top) are examples of high intensity bands. The plasmids contained in the original well are twice the intensity of the insert.

In Lane 5, however, we observed several different bands. This could be due to the extra DNA that was sitting on the top of the well. We decided to re-expose the gel to get better observations on Lanes 3 and 4 because the bands were not as intense. We anticipated seeing a band at about 250 bp because this was the size of the insert (plus extra bp). After re-exposing the gel, we did not find any difference and still did not see any bands in these lanes. We have decided to redo the gel on the part S03511 and so we prepared the master mix with that DNA and incubated it so that we could run the gel tomorrow.

We also cut out the "empty" plasmids from the gel so that we could use this as vectors for the coding sequence of the suppressor tRNAs.

Tomorrow we will be planning out specific tasks for people once the oligos arrive.

==Wednesday, June 10, 2009==

We began this morning by making the agarose gel with the same protocol as yesterday. This time, since we are just re-doing both S03511 inserts, we will have a 1.8% agarose gel.

'''OUTLINE: WHERE DO WE STAND?'''

Yesterday
a. Isolated empty plasmids
b. Verified most inserts
c. Waiting for:
>tRNA oligos
>Primers
a. Will amplify the reporter gene in the plasmid itself

Next?
a. When Primers Arrive
> Amplify the reporter genes with primers (PCR takes ~3 hours)
> Calculate how much reporter plasmid we will need (Start with 50x more than we actually will need)
> Cut amplified sequence (EcoR1 and BamH1/Nco1) then purify what we need
> Cut reporter plasmid (EcoR1 and BamH1/Nco1) then purify what we need
> Ligate amplified sequence and reporter plasmid
b. When tRNA oligos arrive
> Calculate how much of each oligo we will need (takes a while...)
> "Boil and cool" tRNA oligos together
> Ligate fragments (dsDNA) into plasmids we isolated yesterday (~5 minutes)
> Transform the plasmids (~20 minutes)

* LOOK THROUGH LIGATION PROTOCOL (How to assemble genes)
* LOOK THROUGH TRANSFORMATION PROTOCOL

''What we need to get done today'':
1. Prepare the control for the Tet Resistance by BioBricking the part pLac+RBS (S03511) with the TetA (J31007).
2. Check the part number for RFP and also for pLac+RBS+RFP
3. Read through ligation protocol
4. See how many nanograms of plasmid we will need for the tRNAs and for the inserts and multiply this number by 50.
> Check if we have enough plasmids from our mini-prep
5. Purify the plasmids from the gel

After we made this outline, Leland, Olivia and I analyzed the gel we ran this morning and did not see a more than one band in the 2 lanes that contained DNA from S03511 (image below). Since we did not see multiple bands in yesterday's gel either, we conclude that there is something wrong with the DNA rather than with the restriction enzymes that we used to cut it. We concluded that there is probably not an insert in this plasmid both the part and the plasmid are useless.

[[Image: 2009-06-10 10hr 30min.jpg]]

Now, we do not have an immediately accessible promoter. We have decided to order the pBAD promoter from Missouri Western and we will attach the RBS+Tet to this plasmid. Our insert for this will be the larger gene because it is easier to manipulate in the gel than the small gene.

Alyndria Thompson and Romina Clemente are figuring out the size of the plasmids including the reporter genes.

After lunch we performed several steps to isolate and purify the gel to get the empty plasmids will be the vectors for the tRNA DNA. Then, we used the NanoDrop to assess the purity of our DNA. We found that the plasmids were more pure than we had expected. The following values were obtained:

Abs 1.850
A-260 10 mm path 0.172
A-280 10 mm path 0.075
260/280 2.28
260/230 0.09

We then followed the protocol for preparing the agar plates. We put the plate with the agar solution in the Autoclave.

While the solution was in the autoclave, we discussed that we don't really need a promoter in front of RBS+Tet for the gene to be expressed because Tet expression is so strong that the gene would translate without a promoter. However, it would be a good idea to have the promoter because if it was possible to reinforce the translation of the reporter protein, we wanted to do that.

We found that there were some tubes in the lab fridge that contained the pBAD and pLac promoters with RBS. We decided to use these as the promoter and RBS for our experiments. The pLac promoter will be used for RFP and the pBad promoter used with Tet Resistance. We decided to use the pBad with Tet because it is weaker in comparison to pLac. As expression of the Tet gene can be toxic to a cell, we do not want to overexpress the gene with a stronger promoter.

Once the agar solution was sterilized in the Autoclave, we removed it, cooled it, and pipetted the antibiotic resistance into it. We then poured the solution into the plates, removed any bubbles, and let the gel form.

Alyndria Thompson and Romina Clemente concluded that we had more than enough recieving vectors for both the RFP and Tetracycline genes. Since we need 50 nanograms, it is wise to have at least 50 times that much that we can work with, so we need at least 2,500 ng.

RFP 1: 77.6 x 4 = 310.4 ng/uL
x 20 uL = 3,272 ng
RFP 2: 59.5 x 4 = 238 ng/uL
x 20 uL = 4,760 ng
Tet: 40.9 x 4 = 163.6 ng/uL
x 20 uL = 3,272 ng

According to these calculations we have more than 50 times what we need for both instances of RFP and Tet.

Tomorrow, we can prepare the Master Mix for the digestions of both reporter plamids and then add the restriction enzymes once they arrive. Then, we can follow through with the rest of the plans we had made above in the section titles "Next?".

==Thursday, June 11, 2009==

We began this morning by calculating how many uL we would need for each of the mixes for digesting our reporter plasmids. We decided to double the final volume of each mix to 40uL instead of 20uL. Since we knew the concentration of each plasmid and also knew we needed 2500 ng, we cross multiplied to find the uL of DNA we would need for each.

Concentration Calculations:
310.4 ng/1 uL = 2500ng/x uL
x = 8.05 uL

Here is a chart indicating how much of each we used for the 2 mixes.

[[Image: DoubledigestRFPTetR.png]]

On the Promega website, we found a list of compatible buffers for double digestions. Buffers E and H are compatible for the Tet plasmid and RFP plasmid digestions respectively. We fortunately have both buffers in stock.

[[Image:BamEcoR1.png]] [[Image:NcoEcoR1.png]]

We waited for our waited for the restriction enzymes to arrive and in the meantime, we transformed the plasmids with pLab+RBS, pBAd, and pBad+RBS into ''E.coli'' cells. We want the ''E.coli'' cells to replicate the plasmids with these genes because we have a limited amount of genes. [http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_Transformation.html Zippy Plasmid Transformation]

Once our oligos and restriction enzymes arrived, we got to work right away!

'''PCR (Polymerase Chain Reaction)'''

We calculated how much plasmid we will need for the PCR. We decided to dilute the concentration of our plasmids a hundred fold so that we could get pipetable amounts.

''Change in Concentrations''
310.4 ng/uL x 1/100 (dilution factor) = 3.104 ng/uL (RFP)
163.6 ng/uL x 1/100 (dilution factor) = 0.164 ng/uL (Tet)

''Example shown with RFP''
3.104 ng/1 uL = 1ng/ x uL, x = .32
For Tet, x = .61

We also calculated how much of each primer we would need with the following equation:

C1V1 =C2V2
(100 uM) (x) = (1 uM)( 100 uL)
x= 1 uL

[[Image: screen-capture-8.png]]

Each of the 5 of us made had 2 tubes (one for each reporter protein) because there were 5 different forward primers.To each tube, we added the template DNA (either RFP or Tet), water, the respective reverse primer (either RFP or Tet), and the forward primer depending on which 5mer we had chosen. My 5mer was CCAUC.

File:Screen-capture-8.png

2009-06-11T21:10:49Z

ShPunjabi:

IGEM Notebook

2009-06-11T21:10:32Z

ShPunjabi: /* Thursday, June 11, 2009 */

==Wednesday, June 3, 2009==

Romina Clemente and I are trying to find suitable reporter proteins to use. Yesterday, Leland Taylor and Alyndria Thompson were working on ways to insert the gene sequences into the plasmid. Upon seeing how they wanted to manipulated the reporter gene to include the logical clauses, we came up with a few criteria for the reporter genes we would use. The following criteria for genes are listed in order of the broadest aspect to look at to the narrowest aspect:
a) Doesn't contain restriction sites for the 4 restriction enzymes (EcoR1, Xbal, Spel, Pst1) used to cleave the Biobrick part out of the plasmid.
b) Contains 6 cutter restriction sites.
c) These restriction enzymes aren't blunt (cleave straight down at one spot).
d) These restriction sites are close to thge 5' (beginning) end of the sequence.
e) These enzymes are easiest to work with and cheapest.
We are finding the part numbers of the reporter genes we want to use (antibiotic resistance, fluorescence, LacZ) through our own GCAT because we know these ones work. We are then locating these parts on the parts registry [1] website. We copied and pasted the gene sequences we obtained from the registry onto the ApE software [2]. From here, we were able to generate a genetic map of each gene that outlined each restriction site that fit our criteria. We put each genetic map, alongside the part number used, into a Word document.

http://gcat.davidson.edu/GcatWiki/images/0/0e/Restriction_Site_Mapping_on_Reporter_Genes.doc

Later on in the day, we decided that the best way to test for suppression would involve placing the 5mers at the beginning of the reporter gene instead of inserting them into the reporter gene. To do this, we decided that the gene along with its start codon need to be expressed after the 5mer. We will insert a BioBrick plasmid with a 5mer and start codon that will be incorporated before the gene:
RBS-6-8nt-ATG-5mer--ATG-gene

Leland Taylor assembled the oligos that we will need for the BioBrick pieces.

In order to test whether the reporter protein would be expressed regardless of the 5mer being suppressed or not, we would need to remove the suppressor tRNAs from the cell.

Meanwhile, I helped figure out the coding sequence that we will need for the suppressor tRNAs that will bind to the 5mers. We decided that we would be using the following suppressor tRNAs on the Davidson side: CUAGU, CCCUC, CGGUC, CCAUC, and CCACU. We searched through several papers, with little result and finally emailed Dr. Christopher Anderson of UC Berkeley to request the DNA sequence. He emailed us back with a generic DNA code that we could use to create the tRNA; however, for each suppressor tRNA we would use, we would need to change base pairs in the anticodon loop.

The length of the tRNA is 92 nt. The anticodon loop is comprised of 9 nt. Once we have BioBricked the DNA sequence with 4 restriction sites and supplemental nucleotides, the length of the entire gene is 144 nucleotides. This is made up of the 92 nt +22 nt on each side of the sequence (44) + 4 nt on each side of the restriction ends (8).

At the end of the day, we decided to use tetracycline resistance and RFP as our reporter proteins for this tester experiment.

==Thursday, June 4, 2009==

This morning, I began the day by learning how to use the PEARL prgram. Olivia Ho-Shing sat with me and walked me through the program script. And then, Leland Taylor and Shashank Suresh had a problem they wanted us to solve. Leland explained that we need to make sure there is a stop codon in the gene if we fail to suppress the 5mer. The stop codon needs to be before we find another 5mer. They also wanted us to run through both the RFP and Tet Resistance genes to make sure that they did not contain any of the anticodon sequences from our 5 frameshift suppressor tRNAs so as to throw the translation out of frame. They further wanted us to confirm that if we do have successful suppression, there are no stop codons in the middle of the RFP and Tet Resistance genes. PEARL did not find any matches for the suppressor tRNAs within genes. We found 3 matches for stop codons UAA. Two of these occured at the end of the Tet and one at the end of RFP, where they should normally be to end translation into a protein.

Following this, Olivia Ho-Shing and I tried to determine how we can add BioBrick ends to our single stranded DNA sequences that code for the suppressor tRNAs. Our ultimate goal is to find complements for these single strands so they can be put into plasmids. We found the standard prefixes and suffixes for sequences that do not contain ATG (becuase this sequence is a functional RNA and will not be translated) and we placed these before and after the altered 92bp sequence that Dr. Anderson provided us with. The prefixes and suffixes are necessary because they are the extra nucleotides that will be mimicking a "restriction enzyme cutting site". The single strand appeared as below after the Bio Brick prefixes and suffixes were added:

22ntPREFIX--32nt--VARIABLE ANTICODON LOOP--51nt--21ntSUFFIX

We used the suppressor codon CUAGU as our example and placed its appropriate anticodon loop in the sequence. We put this into the lancelator that would give us the other strand as well as the oligos we would need for ideal construction of this double stranded DNA fragment to put into the plasmid. We recieved a total of 4 oligos, 2 of which were variable and 2 of which were constant for all 6 different suppressor tRNAs. Using this double strand, we simulated a restriction enzyme digestion by EcoRI and PstI. We took off the G at both ends of the top strand and then removed the first five nucleotides on each end of the bottom strand. This made the top strand longer than the bottom one. We then proceeded to take the two variable strands and changed the anticodon loop nt for each suppressor tRNA we used.

Around lunchtime, we encountered a problem with the BioBrick scar in the "ATG-5mer subpiece" of the test project. It turns out that after annealing the sticky ends of the Xba1 and Spe1 sites, the scar created had a stop codon TAG (UAG) that was in frame with the suppressed codon. When the Xba1 site on insert anneals to the Spe1 site on the plasmid (the green portions), we get a scar that reads TACTAG.
[[Image:screen-capture.png]]

For the afternoon, Olivia Ho-Shing and I finished assembly of the oligos of the suppressor tRNA codes. We assembled the document below:

http://gcat.davidson.edu/GcatWiki/images/5/5e/TRNAoligostoOrder.doc

We were assigned tasks of creating "cartoons" to illustrate the phenomenon that had occurred. No one could successfully do this however, because we could not understand how the piece had formed. The parts registry website provided wrong information on the restriction site of Spe1 which slowed our efficiency. This project turned into an effort to try to find two other different restriction enzymes that have complementary sticky ends other than Xba1 and Spe1. We want to get rid oc the scar that contains TACTAG.

We ended up deciding that a hybrid of two ideas was needed: restriction enzymes would need to be used to make several reporter gene sequences with different beginnings dependending on the suppressor tRNA sequence needed.

==Friday, June 5, 2009==

Our goal for this morning was to discuss the hybrid PCR approach to assemble the part that comes before the gene we wanted to express. We decided that we would have a BioBrick prefix and a 5mer followed by a sequence of nucleotides whose identity was up to us. Using PCR we would get the second double strand if we have the following:

BioBrick Prefix--ATG--5mer--gene until we get to restriction site

The PCR primer would attach to the ATG and the 5mer and would replicate the complementary base pairs to make a double stranded DNA sequence. As several of us were confused about PCR methods, we looked at the online link provided by Alyndria Thompson's link to the PCR interactive website which was very helpful. We also did some research to discover that the restriction enzyme BseRI (which is contained in the YFP that we wanted to alter) is not compatible with any other restriction enzyme. This is good because we did not want to plasmid containing this gene to close up with Xba1 once we digested it.

The team experienced some confusion with the hybrid idea and spoke to Clif Davis of Missouri Western University on the phone. He told us that there will be one part that is a fragment of the beginning of some nucleotides as a restriction cleaving site, ATG, the logical clause, and the start of the YFP gene that will be placed into a plasmid containing a space. To the left of the space will be the BioBrick Prefix and the right of the space will contain the rest of the genes in the YFP. Therefore, we decided not to alter any BioBrick ends and just look at restriction sites at the beginning of reporter proteins. The small fragment that will be placed into the plasmid will be made by PCR. The PCR will attach a primer onto the ATG and 5mer and replicate.

While we were discussing this, we expanded upon the idea that we can do whatever we want with the "suffix" portion between the 5mer and the beginning of YFP. We decided it would be best to remove ATG and add the first 5 amino acids and the restriction enzyme site. Along with this, if we have the 5mer coding for a certain amino acid (ie methionine) we could replace another codon that codes for methionine WITH that 5mer so long as this codon is contained within the first 15 nt. This way, we would not be altering the protein.

Olivia Ho-Shing came up with an idea of cascading PCR effect for longer logical clauses. Post translational modification of the protein was another one of her ideas. This applies because although we may code for the protein in a given way, the protein may modify or fix itself up to perform a slightly different function in its tertiary form.

Dr. Campbell suggested that we can look up restriction enzymes 400-500 base pairs into the reporter protein we would like to use; however, we have more flexibility in our procedure if we choose more "hardy" cutters. So instead, we could cut with EcoRI instead of with Xba1.

During the afternoon, we set about three tasks:
1) Which primers to use
2) Which promoters to use
3) Which restriction enzymes and how they will determine use of reporter proteins

Olivia Ho-Shing and I began looking up which restriction enzymes were strong so we could base our reporters on that. We began with Tet and RFP since those were the two reporters we had started out with choosing. I found a website that provided enzyme cleavage efficiency based upon the proximity of the site to the end of the PCR fragment that contains it. We discovered the following based upon the attached documents:

http://gcat.davidson.edu/GcatWiki/images/8/8f/Restriction_Enzyme_Cleavage_Efficiency.doc
http://gcat.davidson.edu/GcatWiki/images/9/9a/07Jun09_RE_Cutting_Efficiency_oh.doc

We decided that these two reporters contained efficient enzymes in a good quantity to choose from. On Monday, we will look through these enzymes once more and finalize which enzymes we would like to use with RFP and Tet.

==Sunday, June 7, 2009==

We did preparations for the mini prep this evening. The following notes are courtesy of Olivia Ho-Shing.

1. Sterically transfer ~2mL LB+AMP (AMP are ampicillin resistant plasmids) *Never put fluid back into bottle once it has entered pipette*
2. Use forceps to grab toothpick, scrap some frozen cells, rub in tube with top

'''Tubes'''

S03511 (2)

K091111

B0030

I715039

K091112

I715039

J31007 (2)

B0034

E1010

S03710

==Monday, June 8, 2009==

We reviewed the PCR process this morning as well as the restriction enzyme cleavage process.

Then, Leland Taylor, Olivia Ho-Shing and I all worked on converting the tRNA oligos we made to the template that Missouri Western used. The oligos we had previously made were different from the ones that Dr. Anderson had originally emailed us because further communication between Dr. Anderson and Missouri Western resulted in a better template.

We tried several times to minimize the number of oligos while keeping their individual lengths under 80bp. We managed to do this in the end by creating 7 oligos, 2 of which are variable between the suppressor tRNAs.
We reviewed the following document and finalized the oligos that we needed to order for the frameshift suppressor tRNAs. We decided to keep both the 10bp and 9bp anticodons of CCAUC.

I also looked up the amino acids that corresponded to the FS (frameshift suppressor) tRNAs for future use:

1. CUAGU = Leu
2. CCCUC = Pro
3. CGGUC = Arg
4. CCACU = Pro
5. CCAUC* = Pro
*9 and 10 bp anticodons

Also, we have decided to use NcoI as the restriction enzyme for RFP. This cleavage site occurs 419 bp into the gene. We have decided to use BamHI as the enzyme for Tetracycline Resistance and the cleavage site occurs 290bp into the gene. Both of these enzymes are very powerful and hardy.

We also looked over Missouri Western's tRNA oligos and primer oligos which we approved.
Our oligos were also approved by Missouri Western and Dr. Campbell ordered them while we supervised.

In the afternoon we did the miniprep. The mini prep directions were as appear in the link below.

http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_MiniPrep.html

We then worked with the Nano Prep to dilute our solution of DNA to an absorption of 1.5 and then we made labels for the tubes. The chart below shows relevant information we obtained from the NanoDrop for each of the reporter plasmids.

[[Image: Screen-capture-6.png]]

==Tuesday, June 9, 2009==

'''Preparations for restriction enzymes digestion of plasmids'''

Purpose: To determine the size of the plasmid inserts and ensure that plasmids are what we extracted from the cells during the mini-prep.

Things we need:
DNA ~3uL
Buffer 10x (Want to dilute to 1x) so add 2uL
Enzyme 1 ~1uL
Enzme 2 ~1uL
Water ~13uL
Final Volunter about 20 uL.

Need to choose the right buffer for the restriction enzymes. All buffers come in 10x (10 times more concentrated than what you want it to be when you use it). [http://gcat.davidson.edu/GcatWiki/index.php/Davidson_Missouri_W/Double_Digest_Guide Buffer Website]

Total volume of enzymes has to be 10% or less than final volume (most volume 2uL). Barely submerge the pipet tip into the solution when pipeting the enzymes.
Star (*) activity: Restriction enzyme stops cutting specifically and starts cutting everywhere.

DNA can't be seen by itself so we add EtBr (the bigger the insert, the more will bind) so bigger strands appear brighter. samples.

The order of entry doesn't matter, but make sure to enter the enzymes last.

Will have lots of labeled tubes and will put 3uL of appropriate plasmids into the tubes.
In the meantime, we'll make a "Master Mix" of all the other ingredients in a bigger tube. Always make enough volume for one extra tube.

Enzymes usually have an optimal temperature of 37C. We will let this reaction go for an hour and then we will run it on a gel.

'''Gel Electrophoresis Protocol'''

Website online for ideal gel concentration for resolving different sizes of molecules [http://gcat.davidson.edu/iGEM08/gelwebsite/gelwebsite.html Gel Concentration Website].
See protocol on the Davidson/Missouri Western Wookie for running molecules in the gel and how to digest with restriction enzymes.

'''What's Next?'''

1. 5' end additions
a. Using PCR
b. Cut at the restriction sites EcoR1 and either Bam H1 or Nco1
c. Cut plasmid and reporter with same restriction enzymes as above
d. Run a gel and purify "keeper" DNA (Ligate transform, screen, sequence)
e. Then, we take the "keeper" DNA and combine it with the insert from part

We can have steps d and e ready in advance.

2. tRNAs
a. Assemble the oligos into genes (Requires lots of calculations beforehand, but the actual steps )
b. Plasmids cut up with EcoR1 and Pst1 '''WE CAN USE THE "WASTE" PLASMID FROM THE RESTRICTION ENZYME DIGEST'''
c. Gel purify the plasmids (Ligate transform, screen, sequence)

The following tables include Master Mix measurements, agarose gel concentrations and buffers.
http://gcat.davidson.edu/GcatWiki/images/0/08/Shamitapic3.png
http://gcat.davidson.edu/GcatWiki/images/d/d1/Shamitapic4.png
http://gcat.davidson.edu/GcatWiki/images/6/6f/Shamitapic5.png

We made the Master Mix with our DNA and put it in the incubator at 10:40 AM.

'''Preparing the Gels'''

Now, we will be making 2 gels, 1 at 0.8% and 2.5% (weight to volume percent). The gel volume is 60mL.

'''Definition of 1% solution:
1 gram of agarose/ 100mL buffer'''

So for a 0.8% solution: 0.8 grams agarose/100mL buffer AND we need 60mL (fixed volume for casts run) so multiply that by 0.6.
Final concentration:

.48 grams agarose/60mL buffer

1 uL of EtBr stock for 60mL of gel.
EtBr is in the buffer so it is constantly replenishing itself even though it is moving toward one end in order to make bands on the other end visible.

After Dr. Campbell's demonstration, we each helped prepare a 0.8% buffer solution using the agarose powder, buffer and EtBr. We mixed together the first two substances in a 200mL Erlenmeyer flask and heated it for 120 seconds in the microwave. We then added EtBr and poured the gel solution into the gel cast.

'''Agar Plate'''

After lunch, we grew some more cells for each of the 11 parts in an agar plate labeled "LBAMP-AMC-9 June" so that so would have extra cells that could be stored for up to 2 weeks.
We made small circles on the agar plate and numbered them 1-11. The numbers corresponded to the following parts. We put the plate in the incubator overnight to let the cells grow.

1. B0030 AMC
2. K091111
3. S03710
4. I715039 OEH
5. S03511 ORH
6. I715039 AST
7. B0030 SDP
8. K091112
9. J31007 LJH
10. J31007 REC
11. S03511

'''Unloading the Gels'''

The following shows which parts we entered in which gels:

[[Image: screen-capture-2.png]]

We unloaded the 2.5% agarose first and after observing the bands under a UV light, put the gel into the BioRad machine to observe the bands. The image below is of a Molecular Weight Marker that shows the number of bp in control fragments of DNA according to the distance they travel down the gel.

[[Image: screen-capture-1.png]]

We obtained the following printout for the first gel that was 2.5% Agarose.
[[Image: 2009-06-09_14hr_44min.jpg]]

The bands in the first well at the bottom are those of the molecular weight marker. We skipped the lane above (to the right) of the MWM and entered the rest of the inserts as described in the above table. A light band is visible for each of the 4 other wells. In Lanes 3 and 4 we concluded we are supposed to have approximately 45 bp because the insert is 15bp + 30 bp on either side of the insert (Xba1 and Spe1 restrictions sites + sticky ends + extra bases). We used the same logic for Lanes 5 and 6 which are supposed to contain approximately 86 base pairs.

One discrepancy in this gel, however, is that Lanes 5 and 6, which according to their labels respectively contain the LacIQ promoter and the Lac IQ1 promoter. According to the information online at the parts registry website, pLacIQ1 should be the same length as pLacIQ. The gels do not show this, but instead show that the sample in Lane 5 is smaller than that in Lane 6 because Lane 5 traveled further. Leland Taylor and Romina Clemente sent out an email to Pallavi Penumetcha to ask about this because she had written a paper about this.

We then unloaded the 0.8% agarose gel and then placed this in the BioRad machine under the same protocol as above. The following image is the 0.8% Agarose.

[[Image:2009-06-09_15hr_21min.jpg]]

We observed that the bands in Lane 1 (which is now at the top) are examples of high intensity bands. The plasmids contained in the original well are twice the intensity of the insert.

In Lane 5, however, we observed several different bands. This could be due to the extra DNA that was sitting on the top of the well. We decided to re-expose the gel to get better observations on Lanes 3 and 4 because the bands were not as intense. We anticipated seeing a band at about 250 bp because this was the size of the insert (plus extra bp). After re-exposing the gel, we did not find any difference and still did not see any bands in these lanes. We have decided to redo the gel on the part S03511 and so we prepared the master mix with that DNA and incubated it so that we could run the gel tomorrow.

We also cut out the "empty" plasmids from the gel so that we could use this as vectors for the coding sequence of the suppressor tRNAs.

Tomorrow we will be planning out specific tasks for people once the oligos arrive.

==Wednesday, June 10, 2009==

We began this morning by making the agarose gel with the same protocol as yesterday. This time, since we are just re-doing both S03511 inserts, we will have a 1.8% agarose gel.

'''OUTLINE: WHERE DO WE STAND?'''

Yesterday
a. Isolated empty plasmids
b. Verified most inserts
c. Waiting for:
>tRNA oligos
>Primers
a. Will amplify the reporter gene in the plasmid itself

Next?
a. When Primers Arrive
> Amplify the reporter genes with primers (PCR takes ~3 hours)
> Calculate how much reporter plasmid we will need (Start with 50x more than we actually will need)
> Cut amplified sequence (EcoR1 and BamH1/Nco1) then purify what we need
> Cut reporter plasmid (EcoR1 and BamH1/Nco1) then purify what we need
> Ligate amplified sequence and reporter plasmid
b. When tRNA oligos arrive
> Calculate how much of each oligo we will need (takes a while...)
> "Boil and cool" tRNA oligos together
> Ligate fragments (dsDNA) into plasmids we isolated yesterday (~5 minutes)
> Transform the plasmids (~20 minutes)

* LOOK THROUGH LIGATION PROTOCOL (How to assemble genes)
* LOOK THROUGH TRANSFORMATION PROTOCOL

''What we need to get done today'':
1. Prepare the control for the Tet Resistance by BioBricking the part pLac+RBS (S03511) with the TetA (J31007).
2. Check the part number for RFP and also for pLac+RBS+RFP
3. Read through ligation protocol
4. See how many nanograms of plasmid we will need for the tRNAs and for the inserts and multiply this number by 50.
> Check if we have enough plasmids from our mini-prep
5. Purify the plasmids from the gel

After we made this outline, Leland, Olivia and I analyzed the gel we ran this morning and did not see a more than one band in the 2 lanes that contained DNA from S03511 (image below). Since we did not see multiple bands in yesterday's gel either, we conclude that there is something wrong with the DNA rather than with the restriction enzymes that we used to cut it. We concluded that there is probably not an insert in this plasmid both the part and the plasmid are useless.

[[Image: 2009-06-10 10hr 30min.jpg]]

Now, we do not have an immediately accessible promoter. We have decided to order the pBAD promoter from Missouri Western and we will attach the RBS+Tet to this plasmid. Our insert for this will be the larger gene because it is easier to manipulate in the gel than the small gene.

Alyndria Thompson and Romina Clemente are figuring out the size of the plasmids including the reporter genes.

After lunch we performed several steps to isolate and purify the gel to get the empty plasmids will be the vectors for the tRNA DNA. Then, we used the NanoDrop to assess the purity of our DNA. We found that the plasmids were more pure than we had expected. The following values were obtained:

Abs 1.850
A-260 10 mm path 0.172
A-280 10 mm path 0.075
260/280 2.28
260/230 0.09

We then followed the protocol for preparing the agar plates. We put the plate with the agar solution in the Autoclave.

While the solution was in the autoclave, we discussed that we don't really need a promoter in front of RBS+Tet for the gene to be expressed because Tet expression is so strong that the gene would translate without a promoter. However, it would be a good idea to have the promoter because if it was possible to reinforce the translation of the reporter protein, we wanted to do that.

We found that there were some tubes in the lab fridge that contained the pBAD and pLac promoters with RBS. We decided to use these as the promoter and RBS for our experiments. The pLac promoter will be used for RFP and the pBad promoter used with Tet Resistance. We decided to use the pBad with Tet because it is weaker in comparison to pLac. As expression of the Tet gene can be toxic to a cell, we do not want to overexpress the gene with a stronger promoter.

Once the agar solution was sterilized in the Autoclave, we removed it, cooled it, and pipetted the antibiotic resistance into it. We then poured the solution into the plates, removed any bubbles, and let the gel form.

Alyndria Thompson and Romina Clemente concluded that we had more than enough recieving vectors for both the RFP and Tetracycline genes. Since we need 50 nanograms, it is wise to have at least 50 times that much that we can work with, so we need at least 2,500 ng.

RFP 1: 77.6 x 4 = 310.4 ng/uL
x 20 uL = 3,272 ng
RFP 2: 59.5 x 4 = 238 ng/uL
x 20 uL = 4,760 ng
Tet: 40.9 x 4 = 163.6 ng/uL
x 20 uL = 3,272 ng

According to these calculations we have more than 50 times what we need for both instances of RFP and Tet.

Tomorrow, we can prepare the Master Mix for the digestions of both reporter plamids and then add the restriction enzymes once they arrive. Then, we can follow through with the rest of the plans we had made above in the section titles "Next?".

==Thursday, June 11, 2009==

We began this morning by calculating how many uL we would need for each of the mixes for digesting our reporter plasmids. We decided to double the final volume of each mix to 40uL instead of 20uL. Since we knew the concentration of each plasmid and also knew we needed 2500 ng, we cross multiplied to find the uL of DNA we would need for each.

Concentration Calculations:
310.4 ng/1 uL = 2500ng/x uL
x = 8.05 uL

Here is a chart indicating how much of each we used for the 2 mixes.

[[Image: DoubledigestRFPTetR.png]]

On the Promega website, we found a list of compatible buffers for double digestions. Buffers E and H are compatible for the Tet plasmid and RFP plasmid digestions respectively. We fortunately have both buffers in stock.

[[Image:BamEcoR1.png]] [[Image:NcoEcoR1.png]]

We waited for our waited for the restriction enzymes to arrive and in the meantime, we transformed the plasmids with pLab+RBS, pBAd, and pBad+RBS into ''E.coli'' cells. We want the ''E.coli'' cells to replicate the plasmids with these genes because we have a limited amount of genes. [http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_Transformation.html Zippy Plasmid Transformation]

Once our oligos and restriction enzymes arrived, we got to work right away!

'''PCR (Polymerase Chain Reaction)'''

We calculated how much plasmid we will need for the PCR. We decided to dilute the concentration of our plasmids a hundred fold so that we could get pipetable amounts.

''Change in Concentrations''
310.4 ng/uL x 1/100 (dilution factor) = 3.104 ng/uL (RFP)
163.6 ng/uL x 1/100 (dilution factor) = 0.164 ng/uL (Tet)

''Example shown with RFP''
3.104 ng/1 uL = 1ng/ x uL, x = .32
For Tet, x = .61

We also calculated how much of each primer we would need with the following equation:

C1V1 =C2V2
(100 uM) (x) = (1 uM)( 100 uL)
x= 1 uL

[[Image: screen-capture-8.png]]

IGEM Notebook

2009-06-11T21:09:22Z

ShPunjabi: /* Thursday, June 11, 2009 */

==Wednesday, June 3, 2009==

Romina Clemente and I are trying to find suitable reporter proteins to use. Yesterday, Leland Taylor and Alyndria Thompson were working on ways to insert the gene sequences into the plasmid. Upon seeing how they wanted to manipulated the reporter gene to include the logical clauses, we came up with a few criteria for the reporter genes we would use. The following criteria for genes are listed in order of the broadest aspect to look at to the narrowest aspect:
a) Doesn't contain restriction sites for the 4 restriction enzymes (EcoR1, Xbal, Spel, Pst1) used to cleave the Biobrick part out of the plasmid.
b) Contains 6 cutter restriction sites.
c) These restriction enzymes aren't blunt (cleave straight down at one spot).
d) These restriction sites are close to thge 5' (beginning) end of the sequence.
e) These enzymes are easiest to work with and cheapest.
We are finding the part numbers of the reporter genes we want to use (antibiotic resistance, fluorescence, LacZ) through our own GCAT because we know these ones work. We are then locating these parts on the parts registry [1] website. We copied and pasted the gene sequences we obtained from the registry onto the ApE software [2]. From here, we were able to generate a genetic map of each gene that outlined each restriction site that fit our criteria. We put each genetic map, alongside the part number used, into a Word document.

http://gcat.davidson.edu/GcatWiki/images/0/0e/Restriction_Site_Mapping_on_Reporter_Genes.doc

Later on in the day, we decided that the best way to test for suppression would involve placing the 5mers at the beginning of the reporter gene instead of inserting them into the reporter gene. To do this, we decided that the gene along with its start codon need to be expressed after the 5mer. We will insert a BioBrick plasmid with a 5mer and start codon that will be incorporated before the gene:
RBS-6-8nt-ATG-5mer--ATG-gene

Leland Taylor assembled the oligos that we will need for the BioBrick pieces.

In order to test whether the reporter protein would be expressed regardless of the 5mer being suppressed or not, we would need to remove the suppressor tRNAs from the cell.

Meanwhile, I helped figure out the coding sequence that we will need for the suppressor tRNAs that will bind to the 5mers. We decided that we would be using the following suppressor tRNAs on the Davidson side: CUAGU, CCCUC, CGGUC, CCAUC, and CCACU. We searched through several papers, with little result and finally emailed Dr. Christopher Anderson of UC Berkeley to request the DNA sequence. He emailed us back with a generic DNA code that we could use to create the tRNA; however, for each suppressor tRNA we would use, we would need to change base pairs in the anticodon loop.

The length of the tRNA is 92 nt. The anticodon loop is comprised of 9 nt. Once we have BioBricked the DNA sequence with 4 restriction sites and supplemental nucleotides, the length of the entire gene is 144 nucleotides. This is made up of the 92 nt +22 nt on each side of the sequence (44) + 4 nt on each side of the restriction ends (8).

At the end of the day, we decided to use tetracycline resistance and RFP as our reporter proteins for this tester experiment.

==Thursday, June 4, 2009==

This morning, I began the day by learning how to use the PEARL prgram. Olivia Ho-Shing sat with me and walked me through the program script. And then, Leland Taylor and Shashank Suresh had a problem they wanted us to solve. Leland explained that we need to make sure there is a stop codon in the gene if we fail to suppress the 5mer. The stop codon needs to be before we find another 5mer. They also wanted us to run through both the RFP and Tet Resistance genes to make sure that they did not contain any of the anticodon sequences from our 5 frameshift suppressor tRNAs so as to throw the translation out of frame. They further wanted us to confirm that if we do have successful suppression, there are no stop codons in the middle of the RFP and Tet Resistance genes. PEARL did not find any matches for the suppressor tRNAs within genes. We found 3 matches for stop codons UAA. Two of these occured at the end of the Tet and one at the end of RFP, where they should normally be to end translation into a protein.

Following this, Olivia Ho-Shing and I tried to determine how we can add BioBrick ends to our single stranded DNA sequences that code for the suppressor tRNAs. Our ultimate goal is to find complements for these single strands so they can be put into plasmids. We found the standard prefixes and suffixes for sequences that do not contain ATG (becuase this sequence is a functional RNA and will not be translated) and we placed these before and after the altered 92bp sequence that Dr. Anderson provided us with. The prefixes and suffixes are necessary because they are the extra nucleotides that will be mimicking a "restriction enzyme cutting site". The single strand appeared as below after the Bio Brick prefixes and suffixes were added:

22ntPREFIX--32nt--VARIABLE ANTICODON LOOP--51nt--21ntSUFFIX

We used the suppressor codon CUAGU as our example and placed its appropriate anticodon loop in the sequence. We put this into the lancelator that would give us the other strand as well as the oligos we would need for ideal construction of this double stranded DNA fragment to put into the plasmid. We recieved a total of 4 oligos, 2 of which were variable and 2 of which were constant for all 6 different suppressor tRNAs. Using this double strand, we simulated a restriction enzyme digestion by EcoRI and PstI. We took off the G at both ends of the top strand and then removed the first five nucleotides on each end of the bottom strand. This made the top strand longer than the bottom one. We then proceeded to take the two variable strands and changed the anticodon loop nt for each suppressor tRNA we used.

Around lunchtime, we encountered a problem with the BioBrick scar in the "ATG-5mer subpiece" of the test project. It turns out that after annealing the sticky ends of the Xba1 and Spe1 sites, the scar created had a stop codon TAG (UAG) that was in frame with the suppressed codon. When the Xba1 site on insert anneals to the Spe1 site on the plasmid (the green portions), we get a scar that reads TACTAG.
[[Image:screen-capture.png]]

For the afternoon, Olivia Ho-Shing and I finished assembly of the oligos of the suppressor tRNA codes. We assembled the document below:

http://gcat.davidson.edu/GcatWiki/images/5/5e/TRNAoligostoOrder.doc

We were assigned tasks of creating "cartoons" to illustrate the phenomenon that had occurred. No one could successfully do this however, because we could not understand how the piece had formed. The parts registry website provided wrong information on the restriction site of Spe1 which slowed our efficiency. This project turned into an effort to try to find two other different restriction enzymes that have complementary sticky ends other than Xba1 and Spe1. We want to get rid oc the scar that contains TACTAG.

We ended up deciding that a hybrid of two ideas was needed: restriction enzymes would need to be used to make several reporter gene sequences with different beginnings dependending on the suppressor tRNA sequence needed.

==Friday, June 5, 2009==

Our goal for this morning was to discuss the hybrid PCR approach to assemble the part that comes before the gene we wanted to express. We decided that we would have a BioBrick prefix and a 5mer followed by a sequence of nucleotides whose identity was up to us. Using PCR we would get the second double strand if we have the following:

BioBrick Prefix--ATG--5mer--gene until we get to restriction site

The PCR primer would attach to the ATG and the 5mer and would replicate the complementary base pairs to make a double stranded DNA sequence. As several of us were confused about PCR methods, we looked at the online link provided by Alyndria Thompson's link to the PCR interactive website which was very helpful. We also did some research to discover that the restriction enzyme BseRI (which is contained in the YFP that we wanted to alter) is not compatible with any other restriction enzyme. This is good because we did not want to plasmid containing this gene to close up with Xba1 once we digested it.

The team experienced some confusion with the hybrid idea and spoke to Clif Davis of Missouri Western University on the phone. He told us that there will be one part that is a fragment of the beginning of some nucleotides as a restriction cleaving site, ATG, the logical clause, and the start of the YFP gene that will be placed into a plasmid containing a space. To the left of the space will be the BioBrick Prefix and the right of the space will contain the rest of the genes in the YFP. Therefore, we decided not to alter any BioBrick ends and just look at restriction sites at the beginning of reporter proteins. The small fragment that will be placed into the plasmid will be made by PCR. The PCR will attach a primer onto the ATG and 5mer and replicate.

While we were discussing this, we expanded upon the idea that we can do whatever we want with the "suffix" portion between the 5mer and the beginning of YFP. We decided it would be best to remove ATG and add the first 5 amino acids and the restriction enzyme site. Along with this, if we have the 5mer coding for a certain amino acid (ie methionine) we could replace another codon that codes for methionine WITH that 5mer so long as this codon is contained within the first 15 nt. This way, we would not be altering the protein.

Olivia Ho-Shing came up with an idea of cascading PCR effect for longer logical clauses. Post translational modification of the protein was another one of her ideas. This applies because although we may code for the protein in a given way, the protein may modify or fix itself up to perform a slightly different function in its tertiary form.

Dr. Campbell suggested that we can look up restriction enzymes 400-500 base pairs into the reporter protein we would like to use; however, we have more flexibility in our procedure if we choose more "hardy" cutters. So instead, we could cut with EcoRI instead of with Xba1.

During the afternoon, we set about three tasks:
1) Which primers to use
2) Which promoters to use
3) Which restriction enzymes and how they will determine use of reporter proteins

Olivia Ho-Shing and I began looking up which restriction enzymes were strong so we could base our reporters on that. We began with Tet and RFP since those were the two reporters we had started out with choosing. I found a website that provided enzyme cleavage efficiency based upon the proximity of the site to the end of the PCR fragment that contains it. We discovered the following based upon the attached documents:

http://gcat.davidson.edu/GcatWiki/images/8/8f/Restriction_Enzyme_Cleavage_Efficiency.doc
http://gcat.davidson.edu/GcatWiki/images/9/9a/07Jun09_RE_Cutting_Efficiency_oh.doc

We decided that these two reporters contained efficient enzymes in a good quantity to choose from. On Monday, we will look through these enzymes once more and finalize which enzymes we would like to use with RFP and Tet.

==Sunday, June 7, 2009==

We did preparations for the mini prep this evening. The following notes are courtesy of Olivia Ho-Shing.

1. Sterically transfer ~2mL LB+AMP (AMP are ampicillin resistant plasmids) *Never put fluid back into bottle once it has entered pipette*
2. Use forceps to grab toothpick, scrap some frozen cells, rub in tube with top

'''Tubes'''

S03511 (2)

K091111

B0030

I715039

K091112

I715039

J31007 (2)

B0034

E1010

S03710

==Monday, June 8, 2009==

We reviewed the PCR process this morning as well as the restriction enzyme cleavage process.

Then, Leland Taylor, Olivia Ho-Shing and I all worked on converting the tRNA oligos we made to the template that Missouri Western used. The oligos we had previously made were different from the ones that Dr. Anderson had originally emailed us because further communication between Dr. Anderson and Missouri Western resulted in a better template.

We tried several times to minimize the number of oligos while keeping their individual lengths under 80bp. We managed to do this in the end by creating 7 oligos, 2 of which are variable between the suppressor tRNAs.
We reviewed the following document and finalized the oligos that we needed to order for the frameshift suppressor tRNAs. We decided to keep both the 10bp and 9bp anticodons of CCAUC.

I also looked up the amino acids that corresponded to the FS (frameshift suppressor) tRNAs for future use:

1. CUAGU = Leu
2. CCCUC = Pro
3. CGGUC = Arg
4. CCACU = Pro
5. CCAUC* = Pro
*9 and 10 bp anticodons

Also, we have decided to use NcoI as the restriction enzyme for RFP. This cleavage site occurs 419 bp into the gene. We have decided to use BamHI as the enzyme for Tetracycline Resistance and the cleavage site occurs 290bp into the gene. Both of these enzymes are very powerful and hardy.

We also looked over Missouri Western's tRNA oligos and primer oligos which we approved.
Our oligos were also approved by Missouri Western and Dr. Campbell ordered them while we supervised.

In the afternoon we did the miniprep. The mini prep directions were as appear in the link below.

http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_MiniPrep.html

We then worked with the Nano Prep to dilute our solution of DNA to an absorption of 1.5 and then we made labels for the tubes. The chart below shows relevant information we obtained from the NanoDrop for each of the reporter plasmids.

[[Image: Screen-capture-6.png]]

==Tuesday, June 9, 2009==

'''Preparations for restriction enzymes digestion of plasmids'''

Purpose: To determine the size of the plasmid inserts and ensure that plasmids are what we extracted from the cells during the mini-prep.

Things we need:
DNA ~3uL
Buffer 10x (Want to dilute to 1x) so add 2uL
Enzyme 1 ~1uL
Enzme 2 ~1uL
Water ~13uL
Final Volunter about 20 uL.

Need to choose the right buffer for the restriction enzymes. All buffers come in 10x (10 times more concentrated than what you want it to be when you use it). [http://gcat.davidson.edu/GcatWiki/index.php/Davidson_Missouri_W/Double_Digest_Guide Buffer Website]

Total volume of enzymes has to be 10% or less than final volume (most volume 2uL). Barely submerge the pipet tip into the solution when pipeting the enzymes.
Star (*) activity: Restriction enzyme stops cutting specifically and starts cutting everywhere.

DNA can't be seen by itself so we add EtBr (the bigger the insert, the more will bind) so bigger strands appear brighter. samples.

The order of entry doesn't matter, but make sure to enter the enzymes last.

Will have lots of labeled tubes and will put 3uL of appropriate plasmids into the tubes.
In the meantime, we'll make a "Master Mix" of all the other ingredients in a bigger tube. Always make enough volume for one extra tube.

Enzymes usually have an optimal temperature of 37C. We will let this reaction go for an hour and then we will run it on a gel.

'''Gel Electrophoresis Protocol'''

Website online for ideal gel concentration for resolving different sizes of molecules [http://gcat.davidson.edu/iGEM08/gelwebsite/gelwebsite.html Gel Concentration Website].
See protocol on the Davidson/Missouri Western Wookie for running molecules in the gel and how to digest with restriction enzymes.

'''What's Next?'''

1. 5' end additions
a. Using PCR
b. Cut at the restriction sites EcoR1 and either Bam H1 or Nco1
c. Cut plasmid and reporter with same restriction enzymes as above
d. Run a gel and purify "keeper" DNA (Ligate transform, screen, sequence)
e. Then, we take the "keeper" DNA and combine it with the insert from part

We can have steps d and e ready in advance.

2. tRNAs
a. Assemble the oligos into genes (Requires lots of calculations beforehand, but the actual steps )
b. Plasmids cut up with EcoR1 and Pst1 '''WE CAN USE THE "WASTE" PLASMID FROM THE RESTRICTION ENZYME DIGEST'''
c. Gel purify the plasmids (Ligate transform, screen, sequence)

The following tables include Master Mix measurements, agarose gel concentrations and buffers.
http://gcat.davidson.edu/GcatWiki/images/0/08/Shamitapic3.png
http://gcat.davidson.edu/GcatWiki/images/d/d1/Shamitapic4.png
http://gcat.davidson.edu/GcatWiki/images/6/6f/Shamitapic5.png

We made the Master Mix with our DNA and put it in the incubator at 10:40 AM.

'''Preparing the Gels'''

Now, we will be making 2 gels, 1 at 0.8% and 2.5% (weight to volume percent). The gel volume is 60mL.

'''Definition of 1% solution:
1 gram of agarose/ 100mL buffer'''

So for a 0.8% solution: 0.8 grams agarose/100mL buffer AND we need 60mL (fixed volume for casts run) so multiply that by 0.6.
Final concentration:

.48 grams agarose/60mL buffer

1 uL of EtBr stock for 60mL of gel.
EtBr is in the buffer so it is constantly replenishing itself even though it is moving toward one end in order to make bands on the other end visible.

After Dr. Campbell's demonstration, we each helped prepare a 0.8% buffer solution using the agarose powder, buffer and EtBr. We mixed together the first two substances in a 200mL Erlenmeyer flask and heated it for 120 seconds in the microwave. We then added EtBr and poured the gel solution into the gel cast.

'''Agar Plate'''

After lunch, we grew some more cells for each of the 11 parts in an agar plate labeled "LBAMP-AMC-9 June" so that so would have extra cells that could be stored for up to 2 weeks.
We made small circles on the agar plate and numbered them 1-11. The numbers corresponded to the following parts. We put the plate in the incubator overnight to let the cells grow.

1. B0030 AMC
2. K091111
3. S03710
4. I715039 OEH
5. S03511 ORH
6. I715039 AST
7. B0030 SDP
8. K091112
9. J31007 LJH
10. J31007 REC
11. S03511

'''Unloading the Gels'''

The following shows which parts we entered in which gels:

[[Image: screen-capture-2.png]]

We unloaded the 2.5% agarose first and after observing the bands under a UV light, put the gel into the BioRad machine to observe the bands. The image below is of a Molecular Weight Marker that shows the number of bp in control fragments of DNA according to the distance they travel down the gel.

[[Image: screen-capture-1.png]]

We obtained the following printout for the first gel that was 2.5% Agarose.
[[Image: 2009-06-09_14hr_44min.jpg]]

The bands in the first well at the bottom are those of the molecular weight marker. We skipped the lane above (to the right) of the MWM and entered the rest of the inserts as described in the above table. A light band is visible for each of the 4 other wells. In Lanes 3 and 4 we concluded we are supposed to have approximately 45 bp because the insert is 15bp + 30 bp on either side of the insert (Xba1 and Spe1 restrictions sites + sticky ends + extra bases). We used the same logic for Lanes 5 and 6 which are supposed to contain approximately 86 base pairs.

One discrepancy in this gel, however, is that Lanes 5 and 6, which according to their labels respectively contain the LacIQ promoter and the Lac IQ1 promoter. According to the information online at the parts registry website, pLacIQ1 should be the same length as pLacIQ. The gels do not show this, but instead show that the sample in Lane 5 is smaller than that in Lane 6 because Lane 5 traveled further. Leland Taylor and Romina Clemente sent out an email to Pallavi Penumetcha to ask about this because she had written a paper about this.

We then unloaded the 0.8% agarose gel and then placed this in the BioRad machine under the same protocol as above. The following image is the 0.8% Agarose.

[[Image:2009-06-09_15hr_21min.jpg]]

We observed that the bands in Lane 1 (which is now at the top) are examples of high intensity bands. The plasmids contained in the original well are twice the intensity of the insert.

In Lane 5, however, we observed several different bands. This could be due to the extra DNA that was sitting on the top of the well. We decided to re-expose the gel to get better observations on Lanes 3 and 4 because the bands were not as intense. We anticipated seeing a band at about 250 bp because this was the size of the insert (plus extra bp). After re-exposing the gel, we did not find any difference and still did not see any bands in these lanes. We have decided to redo the gel on the part S03511 and so we prepared the master mix with that DNA and incubated it so that we could run the gel tomorrow.

We also cut out the "empty" plasmids from the gel so that we could use this as vectors for the coding sequence of the suppressor tRNAs.

Tomorrow we will be planning out specific tasks for people once the oligos arrive.

==Wednesday, June 10, 2009==

We began this morning by making the agarose gel with the same protocol as yesterday. This time, since we are just re-doing both S03511 inserts, we will have a 1.8% agarose gel.

'''OUTLINE: WHERE DO WE STAND?'''

Yesterday
a. Isolated empty plasmids
b. Verified most inserts
c. Waiting for:
>tRNA oligos
>Primers
a. Will amplify the reporter gene in the plasmid itself

Next?
a. When Primers Arrive
> Amplify the reporter genes with primers (PCR takes ~3 hours)
> Calculate how much reporter plasmid we will need (Start with 50x more than we actually will need)
> Cut amplified sequence (EcoR1 and BamH1/Nco1) then purify what we need
> Cut reporter plasmid (EcoR1 and BamH1/Nco1) then purify what we need
> Ligate amplified sequence and reporter plasmid
b. When tRNA oligos arrive
> Calculate how much of each oligo we will need (takes a while...)
> "Boil and cool" tRNA oligos together
> Ligate fragments (dsDNA) into plasmids we isolated yesterday (~5 minutes)
> Transform the plasmids (~20 minutes)

* LOOK THROUGH LIGATION PROTOCOL (How to assemble genes)
* LOOK THROUGH TRANSFORMATION PROTOCOL

''What we need to get done today'':
1. Prepare the control for the Tet Resistance by BioBricking the part pLac+RBS (S03511) with the TetA (J31007).
2. Check the part number for RFP and also for pLac+RBS+RFP
3. Read through ligation protocol
4. See how many nanograms of plasmid we will need for the tRNAs and for the inserts and multiply this number by 50.
> Check if we have enough plasmids from our mini-prep
5. Purify the plasmids from the gel

After we made this outline, Leland, Olivia and I analyzed the gel we ran this morning and did not see a more than one band in the 2 lanes that contained DNA from S03511 (image below). Since we did not see multiple bands in yesterday's gel either, we conclude that there is something wrong with the DNA rather than with the restriction enzymes that we used to cut it. We concluded that there is probably not an insert in this plasmid both the part and the plasmid are useless.

[[Image: 2009-06-10 10hr 30min.jpg]]

Now, we do not have an immediately accessible promoter. We have decided to order the pBAD promoter from Missouri Western and we will attach the RBS+Tet to this plasmid. Our insert for this will be the larger gene because it is easier to manipulate in the gel than the small gene.

Alyndria Thompson and Romina Clemente are figuring out the size of the plasmids including the reporter genes.

After lunch we performed several steps to isolate and purify the gel to get the empty plasmids will be the vectors for the tRNA DNA. Then, we used the NanoDrop to assess the purity of our DNA. We found that the plasmids were more pure than we had expected. The following values were obtained:

Abs 1.850
A-260 10 mm path 0.172
A-280 10 mm path 0.075
260/280 2.28
260/230 0.09

We then followed the protocol for preparing the agar plates. We put the plate with the agar solution in the Autoclave.

While the solution was in the autoclave, we discussed that we don't really need a promoter in front of RBS+Tet for the gene to be expressed because Tet expression is so strong that the gene would translate without a promoter. However, it would be a good idea to have the promoter because if it was possible to reinforce the translation of the reporter protein, we wanted to do that.

We found that there were some tubes in the lab fridge that contained the pBAD and pLac promoters with RBS. We decided to use these as the promoter and RBS for our experiments. The pLac promoter will be used for RFP and the pBad promoter used with Tet Resistance. We decided to use the pBad with Tet because it is weaker in comparison to pLac. As expression of the Tet gene can be toxic to a cell, we do not want to overexpress the gene with a stronger promoter.

Once the agar solution was sterilized in the Autoclave, we removed it, cooled it, and pipetted the antibiotic resistance into it. We then poured the solution into the plates, removed any bubbles, and let the gel form.

Alyndria Thompson and Romina Clemente concluded that we had more than enough recieving vectors for both the RFP and Tetracycline genes. Since we need 50 nanograms, it is wise to have at least 50 times that much that we can work with, so we need at least 2,500 ng.

RFP 1: 77.6 x 4 = 310.4 ng/uL
x 20 uL = 3,272 ng
RFP 2: 59.5 x 4 = 238 ng/uL
x 20 uL = 4,760 ng
Tet: 40.9 x 4 = 163.6 ng/uL
x 20 uL = 3,272 ng

According to these calculations we have more than 50 times what we need for both instances of RFP and Tet.

Tomorrow, we can prepare the Master Mix for the digestions of both reporter plamids and then add the restriction enzymes once they arrive. Then, we can follow through with the rest of the plans we had made above in the section titles "Next?".

==Thursday, June 11, 2009==

We began this morning by calculating how many uL we would need for each of the mixes for digesting our reporter plasmids. We decided to double the final volume of each mix to 40uL instead of 20uL. Since we knew the concentration of each plasmid and also knew we needed 2500 ng, we cross multiplied to find the uL of DNA we would need for each.

Concentration Calculations:
310.4 ng/1 uL = 2500ng/x uL
x = 8.05 uL

Here is a chart indicating how much of each we used for the 2 mixes.

[[Image: DoubledigestRFPTetR.png]]

On the Promega website, we found a list of compatible buffers for double digestions. Buffers E and H are compatible for the Tet plasmid and RFP plasmid digestions respectively. We fortunately have both buffers in stock.

[[Image:BamEcoR1.png]] [[Image:NcoEcoR1.png]]

We waited for our waited for the restriction enzymes to arrive and in the meantime, we transformed the plasmids with pLab+RBS, pBAd, and pBad+RBS into ''E.coli'' cells. We want the ''E.coli'' cells to replicate the plasmids with these genes because we have a limited amount of genes. [http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_Transformation.html Zippy Plasmid Transformation]

Once our oligos and restriction enzymes arrived, we got to work right away!

'''PCR (Polymerase Chain Reaction)'''

We calculated how much plasmid we will need for the PCR. We decided to dilute the concentration of our plasmids a hundred fold so that we could get pipetable amounts.

''Change in Concentrations''
310.4 ng/uL x 1/100 (dilution factor) = 3.104 ng/uL (RFP)
163.6 ng/uL x 1/100 (dilution factor) = 0.164 ng/uL (Tet)

''Example shown with RFP''
3.104 ng/1 uL = 1ng/ x uL, x = .32
For Tet, x = .61

We also calculated how much of each primer we would need with the following equation:

C1V1 =C2V2
(100 uM) (x) = (1 uM)( 100 uL)
x= 1 uL

[[Image: screen-capture-7.png]]

File:Screen-capture-7.png

2009-06-11T21:08:45Z

ShPunjabi: