Conclusions

A summary of our project's results and a look ahead to the next step

Takeaways:

Out of the original six constructs, three proved to be most promising: pck overproduced ATP and both petF and petF-pfo overproduced reduced ferredoxin.
Additionally, the two electron donor constructs performed even better in the ΔaceE cells, confirming our modeling predictions.
Finally, we performed a proof of concept experiment by measuring the amount of biotin produced in our electron donor cells, showing that our project could have practical applications.
Our results also indicated that ferredoxin could be a suitable electron donor in E. coli, likely because E. coli has no native regulation for ferredoxin like it does for flavodoxin.

Applications within Nitrogen Project:

Our future directions would be to combine the ATP producing constructs with the reduced electron donor producing constructs to create a particularly super “Super Cell” that can accomplish both ATP and electron donor overproduction. When we started this project, we initially set out to create a suitable environment for nitrogenase activity in E. coli by increasing the amount of intracellular ATP and reduced electron donors. The next logical step would be to get nitrogenase into our “Super Cells” and measure nitrogenase’s ability to fix nitrogen.

Other Applications:

ATP is one of the most useful cellular components and is involved in countless cellular activities. By overproducing ATP, these cellular activities should also be increased. For example, with more ATP, there should be increased recombinant protein production, increased resistance to toxicity, and increased active transport.

Recombinant Protein Production:

When long polypeptide chains are assembled, aminoacyl tRNA synthetases consume ATP¹. We hypothesize that a cell transformed with a plasmid that induces the overproduction of a particular protein would experience an increased demand for ATP. In particular, a leading microbiologist at Intrexon believed that polypeptides with high sulfur concentrations would be particularly energy-consuming and could benefit from increased intracellular ATP levels. We would like to test a reactor in which our cells are allowed to grow to a sizeable population, at which point both ATP and recombinant protein production are concurrently induced.

Electron Donor Application:

One possible application of overproducing ferredoxin is making photosynthesis reactions more efficient. Ferredoxin is the final electron donor in the light-dependent reaction of photosynthesis²; in theory, if the amount of electron donors at this stage increases, the amount of photosynthetic product could increase, improving organism life. This would require further research and experimentation.

References

Overton, Tim W. "Recombinant protein production in bacterial hosts." Drug discovery today 19.5 (2014): 590-601.
Nogales, Juan, et al. "Detailing the optimality of photosynthesis in cyanobacteria through systems biology analysis." Proceedings of the National Academy of Sciences 109.7 (2012): 2678-2683.