The Washington University and Penn State iGEM 2016 project is supported by the Nitrogen Project, a multi-university research initiative to enable plants to fix nitrogen independent of soil bacteria. In previous years, our iGEM team has worked on an intermediate step in this process—transforming E. coli cells (which do not fix nitrogen naturally) with nitrogenase genes from a diazotroph (an organism that fixes nitrogen). This has involved selecting genes from the nif cluster – those known to aid in nitrogenase activity. However, getting these genes from the cyanobacteria Synechocystis into E. coli has proven difficult in practice.

In 2014, the WashU iGEM team reported minimal increases in nitrogenase expression from their nif plasmid. However, these results could not be replicated in 2015. The 2015 iGEM team identified a promising 14 gene subset of the nif cluster but again found E. coli to be an unsuitable host for Synechocystis' nitrogenase. The 2016 WashU / Penn State iGEM team is addressing this problem with a related yet more broadly applicable project.

Nitrogenase activity in E. coli requires a large amount of ATP and electron donors to reduce N2 to consumable nitrates. Our goal for this year’s iGEM is to increase the production of these two cofactors. To do so, we are adding genes that produce electron donors and ATP to plasmids with inducible promoters.

Flavodoxin, an electron donor, is produced naturally in E. coli. We have added fldA, the gene associated with flavodoxin production to a plasmid and used this to transform E. coli. In addition, we created a plasmid containing a gene for ferredoxin, an analogous electron donor found in Synechocystis, from which the nif genes came. Expression of these genes will provide the nitrogenase enzyme with electrons necessary for the reduction of N2, while also helping us to understand the differences between its activity in E. coli and Synechocystis.

Adenosine triphosphate—or ATP—is not just a key factor in the activity of nitrogenase. It is used in active transport, protein synthesis, and many other active processes of E. coli’s metabolism. We have identified 2 genes or gene clusters associated with ATP production: phosphoenolpyruvate kinase (PCK) and phosphoglycerate kinase (PGK), that will allow us to increase the concentration of this molecule within the cell.

Increasing the concentration of intracellular ATP and electron donors may not only be beneficial for nitrogenase activity, but may help facilitate other energetic processes. Manufacturing recombinant proteins, particularly those high in sulfur, require generous amounts of intracellular ATP. Cells with higher ATP concentrations have been shown to be more resistant to toxins, and could therefore be effective in ethanol bioproduction. We plan to continue exploring the possibilities and ramifications of this project.

Additionally, we improved the characterization an HSP BioBrick that is supposed to be activated at high temperatures but had no previous data on the registry. Our characterization experiments, which were repeated by Vilnius iGEM, showed that this HSP was only slightly activated by high temperatures (42C) and its activity greatly decreased over time.

To start following our story, find out how we designed our Super Cells