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Revision as of 03:46, 18 October 2014
Stage 1
Stage 1 of the project deals with testing the cutting efficiency of the Cas9-gRNA complex. For our project, we constructed a library of gRNAs that target an array of genes found in the pathogen Vibrio cholerae. To test whether our library of gRNAs could cut the desired genes, we first began by constructing reporter vectors that have the gRNA binding sequence in the 5' region of our reporter (YFP). When our Cas9 protein is produced in the presence of a tetracycline derivative (doxycycline), formation of the Cas9-gRNA complex can occur, followed by subsequent binding of the reporter vector and cutting of the vector. Once the vector is cut, the bacterial ENZYME, RecBCD nuclease, degrades the linearized vector. The vector also produces the antibiotic cat gene, which offers the bacteria chloramphenicol resistance,so the cells dies once this vector is cut, as we show in the results page for acfA and ctxB gRNA.
Stage 2
Stage 2 of the project deals with testing our library of gRNAs with the Cas9 protein on the endogenous Vibrio cholerae genes. The Cas9 protein should be produced in the presence of a tetratcycline derivative (doxycycline), which can then bind the gRNA of interest, and cut the genome at precise locations. We believe this type of system can offer precision antimicrobial therapeutics against a variety of gastrointestinal pathogens.
We have ordered Vibrio cholerae and will present experimental data from this stage of the project at the iGEM 2014 Giant Jamboree.
Stage 3
Stage 3 of the project begins the second layer of the overall project. This layer requires optimization of the Cas9-gRNA system, and aims at optimizing the transfer of genetic therapeutics among bacterial species. To optimize therapy module transfer, we aim to utilize the M13 phagemid used in previous years to characterize the transfer of our genomic therapy between cells.
Stage 4
Stage 4 of the project aims at continuing the work of stage 3 with the phage that infects Vibrio cholerae, CTX-phi. We hope to engineer packaging sequences and coat proteins from this phage to offer a high level of infection specificity to Vibrio cholerae, while sparing other commensal and symbiotic bacteria.
Stage 5
Stage 5 of the project aims at creating a closed loop of the genetic therapy with a Vibrio cholerae sensor, so that the genetic therapy is turned off in the absence of Vibrio cholerae, but turns on when the pathogenic bacteria invades the gastrointestinal tract.
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Animation showing the proposed mechanism of Stage 1.
