IGEM Notebook
Contents
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.
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
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). 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 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:
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.
We obtained the following printout for the first gel that was 2.5% Agarose.
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.
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.
