Team:WashU StLouis

The production of a bio-fertilizer could benefit the agricultural industry by decreasing the need for energy intensive nitrogen fixation processes. The nif cluster of Cyanothece 51142 consists of 29 genes that construct and regulate a nitrogenase protein complex, which catalyzes the fixation of atmospheric nitrogen. This year, our iGEM team aims to harness the power of nif to produce ammonia in Escherichia coli. After synthesizing a nif- containing plasmid (34 kbp) using the DNA assembler method (Shao et al 2009) and transforming that plasmid into E. coli, our team tested for nitrogenase activity using the acetylene reduction assay. The transformed E. coli were then compared to wild-type under limited nitrogen conditions to check for a competitive advantage. Future tests will evaluate the expression of various nitrogenase subunits, such as nifD and nifK. Our team also aims to further characterize the promoter sequences of the Cyanothece 51142 nif cluster. Between the cysE and nifB genes, there is a 958bp uncharacterized, bidirectional promoter region of particular interest. We are currently using fluorescent reporters to identify key regions within this promoter sequence, and plan to test its function in various environmental conditions.