Team:ColumbiaNYC/Results
Results
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shRNA Design
We chose an shRNA sequenced published on Addgene to knockdown our eGFP, and we compared the shRNA sequence to the eGFP DNA sequence to make sure the knockdown will theoretically occur. To construct the plasmid, we assembled the shRNA-encoding DNA from two segments that made up the sequence because the natural hairpin structure in the shRNA made the full sequence difficult to synthesize in one piece. Because our project design relies on the shRNA to inhibit gene expression (ultimately oncogene expression) and shRNA is not extremely stable (but not unstable), the bacteria must produce large quantities of shRNA for this gene therapy approach to be effective. Thus, we chose to put the gene in a pUC plasmid, which is a very high-copy plasmid (500-600 copies per cell). To increase the production of shRNA further, we placed the shRNA production under the control of a T7 promoter, which causes very strong gene expression compared to other promoters. Our final shRNA plasmid design is the following:
Figure 1: The SilenshR plasmid. This is an IPTG-inducible circuit with a T7 promoter that produces an shRNA that knocks down commercialized eGFP."
Transformation of Plasmid into BL21 (DE3) E.coli
We transformed this plasmid into BL21 (DE3) E. coli cells and got three colonies. To confirm that cloning was successful, we first performed colony PCR to determine if the insert was successfully cloned into the backbone. As can be seen, the colony PCR results show that the insert was successfully cloned in each of the three colonies:
