Team:LaVerne-Leos/Project

Team:LaVerne-Leos 2015.igem.org

Currently, cyanobacteria is used to produce biofuel, however the process is too costly and inefficient to use on a large-scale production to replace fossil fuels. Many researchers have been successful increasing the amount of free fatty acids produced within the cells, which serve as the precursors to biofuel. However, scientists constantly run into a problem: high amounts of free fatty acids are toxic to the cell and kill it. Our aim is to reengineer cyanobacteria to increase the efficiency of biofuel production by improving the yield of fatty acids produced within each cell while solving the toxicity problem. To aid the cells in surviving these high amounts of toxicity we plan to upregulate the cells' carotenoids, which will stabilize their membranes and get rid of reactive oxygen species. With a stronger, more stable cell membrane, the cyanobacteria should be able to produce more free fatty acids, thus producing more biofuel, bringing humanity a step closer to acquiring a source of renewable energy.

Carotenoid pigments are naturally occurring within photosynthetic organisms, and protect the cell against high amounts of light exposure. Specifically, Zeaxanthin is one of the carotenoids that can be synthesized within cells. Some bacteria, such as Staphlycoccus aureus, are able to live in the fatty acids located on our skin (citation). The mechanism in which these bacteria are able to thrive is by upregulating genes related to cell wall thickness and membrane bound carotenoid concentrations. By using this same mechanism, we hope to help the cyanobacteria cope with the fatty acids that they themselves have been genetically modified to make.

Tocopherols are photosynthesis-increasing metabolites that have similar properties to zeaxanthin in cyanobacteria and act as an antioxidant, protecting the cell from lipid peroxidation, a chain reaction affecting unsaturated hydrophobic compounds, including fatty acids, that degrades the cell membrane (Sattler, S. E., Cahoon, E. B., Coughlan, S. J., & DellaPenna, D., 2003). They protect the cell membrane by donating an electron to create resonance-stabilized tocopheroxyl radicals. This stops the lipid peroxidation reaction right in its tracks. Thus, the tocopherols are able to protect polyunsaturated fatty acids from lipid peroxidation, strengthening the cell membrane and possibly preventing cell death. Therefore, we suspect the increased presence of tocopherols could potentially increase a cell's tolerance to fatty acids.



Untransformed bacterial pellet appears white and the bacterial pellet that expresses zeaxanthin appears orange.


Constructs were digested with EcoRI and PstI and run on an agarose gel. The expected band sizes were seen.


GFP Expression in E. Coli. Transformed cells were analyzed by FACS. Mean FL1 signal was calculated. Untransformed E. Coli were used as a control.


The Response of Cell ROX Green and Cell ROX Orange in E. Coli and S. Elongatus. The cells were treated with 50mM H2O2 and analyzed by FACS. Mean FL1 signal was calculated for Cell ROX Green treated cells and mean FL2 signal was calculated for Cell ROX Orange treated cells.

References

Desbois, A. P., & Smith, V. J. (2010). Antibacterial free fatty acids: activities, mechanisms of action and biotechnological potential. Applied Microbiology & Biotechnology, 85(6), 1629–1642. http://doi.org/10.1007/s00253-009-2355-3

Qi, Q., Hao, M., Ng, W., Slater, S. C., Baszis, S. R., Weiss, J. D., & Valentin, H. R. (2005). Application of the synechococcus nirA promoter to establish an inducible expression system for engineering the synechocystis tocopherol pathway. Retrieved from http://aem.asm.org/content/71/10/5678.full

Ruffing, A. M., & Jones, H. D. T. (2012). Physiological Effects of Free Fatty Acid Production in Genetically Engineered Synechococcus elongatus PCC 7942. Biotechnology and Bioengineering, 109(9), 2190–2199. http://doi.org/10.1002/bit.24509

Sattler, S. E., Cahoon, E. B., Coughlan, S. J., & DellaPenna, D. (2003). Characterization of tocopherol cyclases from higher plants and cyanobacteria. Evolutionary implications for tocopherol synthesis and function. Retrieved from www.plantphysiol.org/content/132/4/2184.full

Stahl, W., & Sies, H. (2003). Antioxidant activity of carotenoids. Molecular Aspects of Medicine, 24(6), 345–351. http://doi.org/10.1016/S0098-2997(03)00030-X

About Us

We are the first iGEM team from University of La Verne and La Canada High School. We are an interdisciplinary team with members from biology, math, computer science, art, and library science. We are a community-engaged team that hopes to partner with other high schools, non-profits, and local businesses to create innovative solutions to global issues!

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  • Natural Science Division Office and Biology Department
    1950 3rd St. La Verne, CA 91750

  • Email: ulvigemteam@gmail.com