Collaborative Research: EDGE-FGT: Furthering Progress on a Genetic System for the Oceans' Most Abundant Phototrophs

The picocyanobacterium Prochlorococcus is the smallest, most numerically abundant photosynthesizing organism in the global ocean ecosystem, contributing to almost 10% of global carbon fixation – roughly as much as global croplands. Its abundance is attributed to its enormous genetic diversity; while each cell contains around 2,000 genes, the Prochlorococcus collective has over 80,000 unique genes, allowing these cells to inhabit a wide range of marine environments. Most of these genes are of unknown function, however; thus, researchers are unable to decipher their unique contribution to the ecology of different “ecotypes” along environmental gradients. The bottleneck has been the lack of ability to manipulate the genes in order to determine their function. The investigators will build upon their previous work to optimize a set of methods for the genetic manipulation of Prochlorococcus. Developing these technologies is a critical first step in developing Prochlorococcus as a potential chassis for artificial photosynthesis. Science outreach events at the Boston Public Library focused on cyanobacteria genetics will be designed and implemented, as well as the development of a free, publicly available bioinformatics visualization module to teach students about bacterial horizontal gene transfer events. Recent EDGE funding has allowed the Chisholm and Burton Labs to begin developing a genetic toolkit for Prochlorococcus and its close relative, marine Synechococcus. Because it is unclear which sites in the Prochlorococcus genome will be essential or how efficiently constructs will be integrated into the genome, a two-pronged approach will be utilized in the development of a genetic system in Prochlorococcus. The first aim will involve development of a method for the targeted knockout of genes in the Prochlorococcus chromosome, by either further optimization of the CRISPR-Cpf1 plasmid tool for Prochlorococcus or by induction of the natural-, chemical-, or electro-competent uptake of a linear knockout cassette. The second aim will involve generation of a transposon library in Prochlorococcus, through the use of either natural competence for the uptake an in vitro transposon library, or through generation of an in vivo transposon library by electroporating cells with a pre-assembled custom transposon-Tn5 transposase complex. Collectively, this work will further develop Prochlorococcus as a model system for cross-scales systems biology and allow researchers to characterize how these genes of unknown function contribute to the global success of Prochlorococcus in Earth’s oceans. Results from these studies will be presented at scientific meetings and published in peer-reviewed scientific journals. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.