Difference between revisions of "Part:BBa K3219002"

Revision as of 11:09, 12 October 2019

Plasmid for in vivo expression of dCas9-sgRNA targeting mcyB

This plasmid consists of a cyanobacteria shuttle vector, ribosome binding sites, a dCas9-GFP complex and a sgRNA targeting mcyB of Microcystis Aeruginosa UTEX McyB.

The shuttle vector is from team 2016 iGEM team Nanjing_NFLS, part BBa_K1894001. It consists of two origin 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.

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. The GFP is added to the C-terminus of the dCas9, connected using a linker. There is also a 7x His-Tag, allowing for protein purification.

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 Nermin Adel El Semary and Elke Dittmann.

Sequence and Features