Team:MoWestern Davidson/notebooks

WE NEED TO ADD A SITE MAP

We could try and somehow take the rolling stones' red lips picture and make the tongue green...or something. This could tied to GFP and we need to incorprate an E. coli bacterium into the mix.

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Week 1 (May 25-May 29)

The Missouri Western students came to Davidson to finalize the research agenda for the summer. Both biology and math aspects of the project were discussed in detail. By the end of the week, the team had unified around applications of the Satisfiability (SAT) problem to the genome of E.coli via the use of frameshift suppressor tRNAs.

We would need to design tRNA that would suppress a mutation in a reporter gene that we would also engineer. Therefore, our research would consist of 2 main tracks: tRNA project and the 5mer Reporter Project.

Week 2 (June 1-June 5)

tRNA Project

We began to plan the construction of a 1-SAT model that would include a modified reporter gene suppressed by its respective tRNA.

The two campuses decided to split up the list of tRNAs according to percent suppression. Missouri Western took CUACC, AGGAC, CCAAU, CUAGC, CUACU, and CCACC. Davidson took CUAGU, CCACU, CGGUC, CCCUC, CCAUC-9 and CCAUC-10. We requested the sequence for our tRNAs from Dr. J. Christopher Anderson. After recieving them, we planned their construction via oligo assembly.

By the end of the week, we had planned to use 4 total oligos for our tRNAs.

5mer Reporter Project

In order to minimize major changes made to the protein structures and gene, we decided to place our 5 base pair addition upstream of our reporter protein. We would accomplish this using PCR. We manipulated the PCR primers by designing our forward primer to include ATG, our 5mer, and the first half of the reporter protein up to a restriction site of our choice.

We wanted 1 fluorescence gene and 1 antibiotic resistance gene on each campus. After careful consideration of which reporters had the most "hardy" restriction sites, we decided upon RFP and Tet Resistance on the Davidson campus and GFP and Chlor Resistance on the Missouri campus.

Week 3 (June 8-June 12)

tRNA Project

After editing the first draft of 4 oligos, we devised a tRNA constructed of 7 oligos of shorter lengths, so as to reduce the chance of mutations. We assembled our tRNA from these oligos and ligated/transformed the assembly. By the end of the week, we had obtained colonies from these transformations.

5mer Reporter Project

We chose NcoI as the restriction site in the RFP and BamHI as the site in Tet Resistance. We ordered the oligos of our primers. The forward primer was different for each of the 5 different 5mers on Davidson's campus and 6 different 5mer's on Missouri's campus. We only needed one reverse primer for each reporter gene we used. We carried our PCR using wild type RFP and Tet plasmids as our respective reporter templates.

We only used an aliquot amount to run a gel and verify that the sizes of the PCR fragments included the beginning of the reporter (with ATG and our 5mer) to the restriction site we chose.