Team:Nevada

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A Cooperative Relationship between Cyanobacteria and E.Coli
for production of Biofuels

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 (Galbe et al., 2007). 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.

 NEWS UPDATES

After Months of Trial and Error, Production of Ethanol in E coli is Possible!

     One of the major goals of the Nevada 2011 project is to synthesize ethanol in E. coli, a bacteria well characterized by scientists. In order to produce ethanol, two enzymes need to be produced in E. coli; pyruvate dehydrogenase (PDH) and alcohol dehydrogenase (ADH). Jovanna Casas and her E. coli project team members transformed E. coli with these ethanol genes under the control of a sigma 70 constitutive promoter from the iGEM registry (J23101). Now all they had to do was assay for enzymatic activity. Sounds simple, but this was where the real challenge began for the Nevada Team.

     Casey Lear and her enzymology team were responsible for developing a classic alcohol dehydrogenase enzymatic assay, but according to Casey, “Only when high concentrations of ethanol were added to the assay was there any absorbance correlating to ADH activity.” The enzymology group concluded that the ADH assay they developed was not sensitive enough to detect ethanol in the low concentration ranges needed by Jovanna’s E. coli group.

     The E. coli group opted to use a more sensitive alcohol oxidase enzyme instead. The team purchased an EnzyChrom Ethanol Detection Kit (alcohol oxidase) from BioAssay Systems to test ethanol production in their sigma70/PDC/ADH operon transformed into the NEB Iq E.coli cell line. “We grew the cultures with the addition of 2% glucose, since glucose can help improve E. coli growth. With this new assay, we were able to detect 0.02% ethanol”.

     “To further improve ethanol production, we contacted UNIPV-Pavia, who in 2009 was able to attain 3% ethanol production. UNIPV-Pavia used 10% glucose and induced cultures with 1% ethanol to stimulate ADH activity.” Jovanna and her team grew the cultures in 10% glucose and induced with 1% ethanol, yielding 0.018% ethanol.
Can We Make Biofuels? Yes We Can!

     Megan Tabor and her group of top notch researchers have engineered E. coli with Bay Laurel Thioesterase (BTE) under the control of the sigma 70 constitutive promoter to produce fatty acids. The team has shown that E. coli secretes 100-200uM fatty acids into the surrounding media in NEB Beta cells.

     When the group first received these results, they were ecstatic, but they knew that there was still hard work ahead. It was important for the team to show that not only were they producing larger amounts of fatty acids, but also fatty acids that were 12 carbons in length, instead of the usual 16 and 18 carbon chains found in the typical cooking oil. “Medium chain fatty acids, such as C-12 fatty acids, can be used not only as a biofuel for automobiles, but can also be used as a jet fuel” stated Jovanna Casas, a fellow researcher and close colleague. The group is in the process of repeating their work using chromatography to determine the size of the fatty acids and also testing different E. coli strains to determine the optimal strain for producing fatty acids.
Growing Cyanobacteria and E. coli together, can it be done? Scientists may soon have an answer.

     The co-culturing apparatus has just been delivered by Matt Bowden the team’s lead mechanical engineer. The device will allow for growth of E. coli fed by Synechocystis without cross-contamination between the chambers. Stress testing of the device can now begin, in order to determine how to effectively keep it sterile, prevent all leaks, and ensure the water pump can run for the durations needed to culture cyanobacteria.

      The most important of these tests will be to show that we can prevent all E. coli from entering the cyanobacteria chamber, as contamination will quickly smother the cyanobacteria and prevent photosynthesis. Methods will also need to be developed to sterilize the apparatus, as it is not autoclavable in its entirety and cannot be easily assembled in the sterile hoods available to the team. This may involve running the device while pumping bleach through the system followed by a pumping with sterile water. However, it is unknown if all of the tubing will be resistant to this treatment, and other sterilizing chemicals may need to be used. Finally, we need to be sure that our water pump can run for several days at a time without burning out. If the pump cannot last for the time required for Synechocystis to grow, we will need to redesign the apparatus with a sufficient water pump.

      Once stress testing has been concluded, we can move on to the final test of our project, in which we will grow engineered E. coli to produce biofuels from sugars provided by our Synechocystis.

A poster of our project can be found by Clicking here.


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