Team:Nevada

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Team Nevada 2011 Abstract:

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

Testing Ethanol
AGP/inv Operon
Apparatus

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.

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.

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

@@ Line 35: / Line 35: @@
                                  <div class="col-one col">
 <div class="abstractdesc">
-<u>Pyruvate Decarboxylase & Alcohol Dehydrogenase coding regions under Sigma 70 Constitutive Promoter (J23101)</u>
+<u>After Months of Trial and Error, Production of Ethanol in E coli is Possible!</u>
 <br><br>
-      We first tested ethanol production in s70/PDC/ADH in NEB B10 E. coli cells using EnzyChrom Ethanol Detection Kit (BioAssay Systems).  20 samples were tested, however no samples yielded absorbance over background.
+      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.
-<br><br>     An ADH enzymatic assay was performed by the Enzymology team.  Results were negative as ethanol standards of low concentrations (<20µM) were not detected. Only when high concentrations of ethanol were added was there any absorbance correlating to ADH activity.  We concluded that the ADH enzyme was not sensitive enough to detect ethanol within the concentration ranges we were expecting, and opted to use a more sensitive Alcohol Oxidase enzyme instead.
+<br><br>     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.
-<br><br>     To test the presence of PDC activity, Aldehyde Detection plates were created.  When an aldehyde is present, a bright pink to red colony will form after 2 hours of incubation.  (insert original picture from article here).  After streaking s70/PDC/ADH in NEB B10 E. coli cells onto the plates, we yielded pink colonies after 2 hours of incubation which intensified after overnight incubation (insert s70/PDC/ADH in NEB B10 picture).
+<br><br>     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”.
 </div>
                                  </div>
                                  <div class="col-two col">
 <div class="abstractdesc">
-<br><br>     To once again test ADH activity, we transformed s70/PDC/ADH into NEB High Expression Iq cells.  These cells were plated on the aldehyde detection plates and demonstrated a more intense pink colony than the NEB B10 cells. (insert s70/PDC/ADH in NEB Iq picture).
+<br><br>     “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!
-<br><br>       The EnzyChrom Ethanol Detection Kit to test ethanol production in s70/PDC/ADH in NEB Iq E. coli cells.  Here, we grew the cultures with the addition of 2% glucose, since glucose can help improve E. coli growth.  With these constructs, we were able to detect 0.02% Ethanol.  (insert graph)
-<br><br>       To further improve ethanol production, we contacted UniPavia, who in 2009 was able to attain 3% ethanol production.  They used 10% glucose and induced cultures with 1% ethanol (to improve ADH activity).
 </div>
                                  </div>