Team:HK SSC/Design
Design
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 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.
Fig1. BBa_K1894001 shuttle vector (Restriction enzyme sites and linker not shown) (photo from snapgene)
B. dCas9-GFP
A green fluorescence protein was added to the C-terminus of the dCas9 enzyme, connected using a linker. This allows visual confirmation of successful transformation and indicates that the dCas9 enzyme has been expressed. A ribosome binding site was added to this construct. The stop codon of GFP was mutated in order to allow for continuous expression. There is also a His-Tag to allow for protein purification. The dCas9 construct was adopted from
Fig. 2 dCas9-GFP construct (Restriction enzyme sites and linker not shown) (photo from snapgene)
4. sgRNA
The sgRNA targets 25 base pairs on the mcyB gene of Microcystis Aeruginosa UTEX 2388.
Fig 3. sgRNA design (Restriction enzyme sites and linker not shown) (photo from snapgene)
After various restriction enzyme cuts and ligation, we have a final product of 12, 371 base pairs.
Fig 4. The final plasmid after assembling the 3 parts using restriction enzyme cuts and ligation.