Team:HK SSC/Design
Design
Expression of dCas9-sgRNA-GFP Complex for gene silencing
We designed a plasmid that can be expressed in both E.coli cells and Microcystis Aeruginosa cells, encoding dCas9, green fluorescence protein and an sgRNA targeting McyB.
Our plasmid is cloned by assembling 3 parts: the shuttle vector, the dCas9-GFP complex and the sgRNA.
A. Shuttle vector
The shuttle vector we used is from the iGEM part registry BBa_K1894001 (hyperlink http://parts.igem.org/Part:BBa_K1894001) from iGEM team Nanjing_NFLS. It is a shuttle vector that can replicate in E.coli and cyanobacteria. It consists of a T7 promoter and a CaMV 35S RNA promoter , allowing dCas9-gfp and sgRNA expression in both E.coli and cyanobacteria. Besides, it also confers Kanamycin resistance for selection. We divided the plasmid into 3 fragments, and assembled them using Gibson Assembly. Our Sanger sequencing results have shown no mutations in the junctions.
