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| − | Microcystis aeruginosa is one of the most common species responsible for harmful algal blooms. This cyanobacterium is capable of producing microcystin, a hepatotoxin that could damage the liver, and is regulated by the United States Environmental Protection Agency. However, Microcystis aeruginosa holds its ecological value of heavy metal sorption and oxygen synthesis. In this project, 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 guide RNA(sgRNA), the dCAs9-sgRNA complex specifically binds to the McyI gene and blocks transcript elongation, leading to the repression of the McyI gene without altering the chromosome of the Microcystis. This system is commonly known as CRISPRi. 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, oxygen synthesis rate and metal sorption rate of Microcystis. | + | |
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