IGEM 2015 Project Description.
The 2014 WashU iGEM team successfully transferred the nif cluster from Cyanothece 51142 to E. coli. However, the nitrogenase activity they measured was minimal. Therefore, the WashU/Penn State 2015 team dedicated itself to increasing the nitrogenase activity of last year’s E. coli strain by knocking down and overexpressing certain genes on the nif-containing plasmid. Additionally, we are using genome-scale metabolic modeling to guide potential genetic interventions and facilitate nitrogen fixation in E. coli. We are performing a systematic analysis of metabolites and genes in our model to optimize nitrogenase activity.
A few weeks into our work, a member of our lab had trouble repeating the 2014 results and we began to consider the notion that E. coli may be incompatible with components of the plasmid—whose parts come directly from Cyanothece 51142—at the transcriptional, translational, or post-translational levels. After an evaluation of our remaining time and resources, we decided to construct a more minimal nif cluster for E. coli in order to reduce the effects of any native regulation present in the original plasmid we used. We plan to continue our analysis of the nif cluster using overexpression, knockdown, and metabolic engineering.
Our team is part of a larger project that is seeking to confer nitrogen fixation capabilities to a non-diazotrophic cyanobacterium, Synechocystis pp. 6803. Synechocystis is evolutionarily related by the endosymbiotic theory to plant chloroplasts, so a nitrogen-fixing Synechocystis could be used as a model to engineer plants that fix their own nitrogen. We hope that the results of this year’s iGEM team will be useful to the researchers working on The Nitrogen Project since E. coli is a well-characterized, fast-growing model organism.