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Team:MoWestern Davidson/project design
From 2009.igem.org
Project Design / Approaches
Continuum (scale) FSL Design/tRNAS
To solve the SAT problem, we constructed reporter genes that express a phenotype in the event E. coli solves the SAT problem. To design these reporter genes, we constructed a continuum to model the different level of bacterial automation in solving the SAT problem. This enabled us to select the level of automation we desired and design reporter genes based off of that level. The basic concept behind the reporter genes involves inserting a 5 base pair insertion. This insertion serves to throw off the reading frame in translation, causing a nonsense protein. We then provide the cells with different suppressor tRNAs that may or may not suppress this insertion. If the insertion is suppressed, the reading frame will be restored and the protein will be expressed. These suppressors represent the inputs of the SAT problem, and the 5 base pair insertions represent the clauses of the problem.
Designing the Locks and Keys:
We employed two separate approaches to design and construct our suppressor tRNAs (keys) and the frameshifted reporter proteins (locks). The genomic sequence of the novel five nucleotide anticodon tRNAs were obtained from papers by Thomas J. Magliery, J. Christopher Anderson and Peter G. Schultz {Magliery et. al. & Anderson et. al.}. We used synthetic oligo nucleotides to assemble the twelve tRNAs with flanking biobrick prefix and suffix sticky ends. Alternately, the mutated reporter genes were designed and constructed by PCR directed mutagenesis.
