Team:Nevada

From 2011.igem.org

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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.
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<u>Growing Cyanobacteria and E. coli together, can it be done? Scientists may soon have an answer.</u>
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Co-cultivation is a process where two different species are grown together in one environment. In the case of the Nevada 2011 iGEM team, they are trying to co-cultivate two very different bacteria in one growth chamber apparatus. The first bacteria, E. coli, is a well-studied laboratory bacteria that can be genetically engineered to produce all kinds of high value products, such as pharmaceuticals and biofuels. The second bacteria, Cyanobacteria (Synechocystis), is capable of undergoing photosynthesis, a process of turning sunlight into sugar. The rationale behind creating a co-cultivation apparatus is to grow a genetically engineered version of Synechocystis that secretes sugar (glucose) into the surrounding environment and feeds the genetically engineered E.coli cells. Since 30-40% of the total costs of growing genetically engineered E.coli is from feeding them the nutrients such as glucose, the Nevada team is finding a way to lower the cost of production of biofuels and other genetically engineered industrial products by having the Synechocystis feed the E. coli so scientists won’t have to. Work on this project is now underway.
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  <br>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.<br>
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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.<br>
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The co-cultultivation apparatus has just been delivered by Matt Bowden the team’s lead mechanical engineer.  The device will allow for the growth of E. coli fed by Synechocystis without cross-contamination between the chambers. Stress testing of the device is now taking place in order to determine how to effectively keep the chambers sterile, prevent all leaks, and ensure that 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” states Bryson Wheeler, a team member heavily involved in media optimization and a team member of the co-cultivation project.   “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, they can move on to the final test of the project, growing engineered E. coli that produce biofuels generated from sugars provided by our Synechocystis.<br>
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  Optimal media constituents are being tested to determine the optimal growing conditions for both cyanobacteria and E. coli.<br>
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  <br><font size="3px !important">A poster of our project can be found by  
  <br><font size="3px !important">A poster of our project can be found by  
<a href="https://static.igem.org/mediawiki/2011/2/2e/UNR_Poster.png" style="color:White;">Clicking here.</a></font>
<a href="https://static.igem.org/mediawiki/2011/2/2e/UNR_Poster.png" style="color:White;">Clicking here.</a></font>

Revision as of 16:13, 26 September 2011



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. 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 hope to be able to test out this new procedure before the iGEM Competition on October 8, 2011. More results to come!


Currently, each gene "part" of the AGP knockout/inv Operon has been successfully amplified and isolated by PCR. Gel analysis of each part indicates that the genes have been reconstructed to include 20 bp overlap regions with the adjacent genes in the construct design, as depicted in the Project Overview. Initially, the overlap regions were created with the intent of assembling the construct by Gibson assembly, or similarly by SLIC. Due to the complexity of the construct design, alternative protocols are currently being explored. PCR strategies are under investigation to create "bridges" between adjacent parts using the primers previously designated for Gibson assembly.
Growing Cyanobacteria and E. coli together, can it be done? Scientists may soon have an answer.

Co-cultivation is a process where two different species are grown together in one environment. In the case of the Nevada 2011 iGEM team, they are trying to co-cultivate two very different bacteria in one growth chamber apparatus. The first bacteria, E. coli, is a well-studied laboratory bacteria that can be genetically engineered to produce all kinds of high value products, such as pharmaceuticals and biofuels. The second bacteria, Cyanobacteria (Synechocystis), is capable of undergoing photosynthesis, a process of turning sunlight into sugar. The rationale behind creating a co-cultivation apparatus is to grow a genetically engineered version of Synechocystis that secretes sugar (glucose) into the surrounding environment and feeds the genetically engineered E.coli cells. Since 30-40% of the total costs of growing genetically engineered E.coli is from feeding them the nutrients such as glucose, the Nevada team is finding a way to lower the cost of production of biofuels and other genetically engineered industrial products by having the Synechocystis feed the E. coli so scientists won’t have to. Work on this project is now underway.



The co-cultultivation apparatus has just been delivered by Matt Bowden the team’s lead mechanical engineer. The device will allow for the growth of E. coli fed by Synechocystis without cross-contamination between the chambers. Stress testing of the device is now taking place in order to determine how to effectively keep the chambers sterile, prevent all leaks, and ensure that 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” states Bryson Wheeler, a team member heavily involved in media optimization and a team member of the co-cultivation project. “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, they can move on to the final test of the project, growing engineered E. coli that produce biofuels generated from sugars provided by our Synechocystis.

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


SPONSORS

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