IGEM Notebook

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:

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.

June 7, 2009

We did preparations for the mini prep this evening.

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

<b/>Tubes<b/> S03511 (2) K091111 B0030 I715039 K091112 I715039 J31007 (2) B0034 E1010 S03710