Microcystis Aeruginosa is a noxious cyanobacterium that produces a hepatotoxin, Microcystin. This plasmid silences the McyB gene of Microcystis Aeruginosa UTEX2388 using the dCas9 and sgRNA. According to previous researchers, Microcystin cannot be produced without McyB[1]
This plasmid can be used for in vivo or in vitro expression of dCas9 and sgRNA. This plasmid consists of a cyanobacteria shuttle vector, ribosome binding sites, a dCas9-GFP complex and a sgRNA targeting McyB of Microcystis Aeruginosa UTEX 2388 McyB.

Shuttle Vector

The shuttle vector is from team 2016 iGEM team Nanjing_NFLS[2], part BBa_K1894001. It consists of two origins of replications, f1 ori and pUC ori, which allows it to replicate in both E.coli cells and cyanobacteria cells. It also consists of a t7 promoter, CaMV35S promoter, and Kanamycin resistance gene. The T7 promoter allows expression of the dCas9-GFP-sgRNA in E.coli and the CaMVS promoter allows expression in cyanobacteria.

dCas9

The dCas9, also known as a catalytically dead Cas9 enzyme, is a mutated Cas9 enzyme without endonuclease activity. With the help of a single-guide RNA (sgRNA), it specifically binds to the target sequences and blocks transcript elongation by RNA polymerase. In our project, the dCas9 construct was from the iGEM part registry BBa_K1689013, by teamiGEM15_Peking[3]. The GFP is added to the C-terminus of the dCas9, connected using a linker. This is to allow visual confirmation of successful transformation and indicates that the dCas9 enzyme has been expressed. There is also a 7x His-Tag, allowing for protein purification.

sgRNA

The sgRNA consists of a handle, a base-pairing region and a terminator. The base-pairing region is designed to target 25 base pairs of the McyB gene of Microcystis Aeruginosa UTEX2388.

Usage

This part can be used by transforming it directly into Microcystis Aeruginosa UTEX 2388 via electroporation or naturual transformation. Our team used the protocols provided by Au Yang Qin [4], Nermin Adel El Semary[5] and Elke Dittmann[6]. After the expression of dCas9 and sgRNA will result in the repression of the McyB gene. Thus, no Microcystin will be produced.

Team:HK SSC/Composite Part

Composite Parts

Plasmid for in vivo expression of dCas9-sgRNA targeting mcyB