Santa Cruz is surrounded by agricultural communities dependent on local freshwater sources. We found inspiration for our iGEM project 16 miles south in Watsonville, which is home to Pinto Lake. For decades, Pinto Lake has been contaminated with microcystin, an environmentally-harmful hepatotoxin known to cause nausea, vomiting, diarrhea, pneumonia, and in severe cases, liver failure and death. With Watsonville’s location in the heart of leafy-green production, there were concerns about accumulation of microcystin in crops, livestock, and water, halting the use of Pinto Lake as an agricultural freshwater source. Pinto Lake remained as a recreational water body, but rising microcystin levels threaten this utility as well.
After repeated lake closures due to concerns for public recreational safety, with the most recent being August 6, 2023, the City of Watsonville is in a position of being unable to afford expensive treatments while blooms are back in full force. The need for an accessible, effective, and ecologically-conscious solution to dangerous microcystin levels is what inspired our project: Toxic Algal Bloom Interference (TABI).
Our initial approach involved interfering with toxic gene expression in Microcystis aeruginosa (M. aeruginosa), the cyanobacteria that is responsible for microcystin production. We planned to design a long-term treatment using a plasmid that halts expression of microcystin synthetase genes in M. aeruginosa. Due to the difficulty of transforming cyanobacteria like M. aeruginosa, however, we redirected our efforts on developing a method that improves transformation efficiency in M. aeruginosa and other non-model organisms.
We aim to improve transformation efficiency in M. aeruginosa by using the Stealth program to identify underrepresented R-M motifs in the M. aeruginosa genome and by using the Chameleon project made by our team to remove motifs from CDSs via synonymous codon-optimization of a broad host range plasmid (pSPDY). The resulting improvement in transformation efficiency will be assessed to validate Stealth and the Chameleon project. These bioinformatic tools and our experiments will establish a method for preparing plasmids for efficient transformation into M. aeruginosa, which we will leverage for efficient toxin disruption in the future. Beyond the M. aeruginosa context, this project will also provide a foundation for engineering other non-model species.