Difference between revisions of "IGEM 2009 notebook"

Revision as of 15:01, 8 June 2009

Roclemente 09:57, 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 [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

Roclemente 16:54, 3 June 2009 (EDT)

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 [3]. 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.

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.

--Roclemente 12:06, 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.

--Roclemente 17:13, 5 June 2009 (EDT)

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

--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:

This is the reverse primer we will order: