Team:Nevada

In light of the growing energy crisis, much research has been devoted to finding economical means of producing renewable fuels.
Traditional methods for obtaining biofuels have relied mainly on the fermentation of agricultural crops. However, there are a number of problems with this approach: the reduction in land available for food production, relatively low levels of CO2 biofixation, and large biomass requirements. Our project aims to overcome these problems by utilizing E. coli for the production of biodiesel (C-12 fatty acids) and bioethanol. In the past there have been a number of examples of biofuel production in E. coli; however 30-40% of production cost is based on media costs. Our project will surmount these high production costs by engineering the cyanobacteria, Synechocystis PCC 6803, to secrete large quantities of glucose that will feed our biofuel-producing E. coli. Cyanobacteria and E. coli will be co-cultivated in an apparatus that allows for the mutual transfer of carbon to produce biofuels. Not only will this project provide an efficient means for producing biofuels without the need for a carbon source, but it will also create a novel cooperative system between bacterial species that may have further industrial implications.

E. coli will be pumped through the Cyanobacteria chamber in dialysis tubing to allow for medium exchange. Florescent lights will be installed for Cyanobacteria to carry out photosynthesis. Air will be pumped to both cylinders for the growth of bacteria. Samples of the media will be taken from each cylinder to test for the production of sucrose, fructose, ethanol, and fatty acids.

The main genes necessary to assemble our two constructs, agp and ThiE, have been successfully isolated from the genomic DNA of Synechocystis pcc 6803. Our promoter, petBD, as well as KnR, the gene for kanamycin resistance have also been successfully isolated and are now ready for Gibson assembly.
The Bay Laurel thioesterase, and Z. mobilis pyruvate decarboxylase/alcohol dehydrogenase coding regions were cloned behind the Sigma 70 constitutive promoter. Assays are being developed and tested for protein activity.

Optimal media constituents are being tested to determine the optimal growing conditions for both cyanobacteria and E. coli.