Expression of dCas9-sgRNA Complex in Microcystis Aeruginosa Resulting in the Repression of its Toxin-producing Gene
Microcystis aeruginosa is one of the most common cyanobacteria responsible for harmful algal blooms. This cyanobacterium produces microcystin, a hepatotoxin that damages the liver. However, direct lysis of Microcystis aeruginosa may not best for the environment as it holds ecological values of heavy metal sorption and oxygen synthesis. We hope to silence the microcystin biosynthesis cluster(mcy) using a catalytically dead Cas9 (dCas9) enzyme lacking endonuclease activity. When the dCas9 enzyme is co-expressed with a single guide RNA(sgRNA), the dCas9-sgRNA complex specifically binds to the McyB gene and blocks transcript elongation, leading to the repression of the McyB gene without altering the chromosome of the Microcystis. Here we provide the design of a dCas9-sgRNA expression gene in a shuttle vector that can replicate in both E.coli and cyanobacteria. We will also be conducting downstream analysis to see how our dCas9-sgRNA expression plasmid affects the microcystin-production rate and oxygen synthesis rate of Microcystis.
We are a team of high school students aged 14-17 from St. Stephen’s College Hong Kong. We are all passionate about synthetic biology. Through synthetic biology, we hope to make a difference to the current world, and to share the joy of biology. Throughout this year, we have fought hard for this project, conducting numerous laboratory sessions and paperwork. We look forward to presenting our project to the world in the iGEM Giant Jamboree.