Team:GeorgiaTech/Project

From 2014.igem.org

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<a href="#Results" style="font: 18px 'Bitter' serif; color: #840000;">Results</a>
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<a href="#Status" style="font: 18px 'Bitter' serif; color: #840000;">Project Status</a>
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<h2> Results </h2>
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<h2> Project Status </h2>
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<p><b>The Biobricked sMMO genes</b> are currently having RBS and promoter pairs of varying strengths inserted into the plasmids, with plans to characterize their different expression levels via 2D-SDS PAGE.</p>
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<p><b>Currently,</b> RBS's are being inserted into the sMMOX and sMMOY biobricks, while all four RBS and promoter pair crosses have already been created using sMMOZ and sMMOB. sMMOC has both high and low RBS inserted along with being ligated back into the standard vector and is in the process of promoter insertion.</p>
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<h3>Current Confirmed RBS, Promoter, sMMO Gene Biobricks
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<th>sMMO Gene</th><th>HE RBS (promoterless)</th><th>LE RBS (promoterless)</th><th>HE Promoter/HE RBS</th><th>HE Promoter/LE RBS</th><th>LE Promoter/HE RBS</th><th>LE Promoter/LE RBS</th>
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<tr><td> Z <td> √ <td> √ <td> √ <td> √ <td> √ <td>
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<tr><td> B <td> √ <td> √ <td> √ <td> √ <td> √ <td>
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<tr><td> C <td> √ <td> √ <td>  <td>  <td>  <td>
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Revision as of 22:45, 15 October 2014

RBS Cloning Using PCR Primers: 100%

Promoter Cloning Using PCR Primers: 100%

Creation of sMMO Expression Vectors: 80%

Characterization of Recombinant sMMO Proteins: 0%

What the Frack?

Fracking is a mining technique in which a high pressure fluid is injected into a well bore in order to generate small fractures. These fractures allow previously inaccessible hydrocarbons to migrate to the well for later extraction. Because fracking can be applied to most oil and natural gas wells, it is a common technique for extending the life of these wells, and thus lowering the need for construction of new wells. While fracking activity is crucial for providing low cost energy resources for American industry, there is a difficulty in storing and treating fracking waste water in an environmentally and economically suitable fashion.

The waste water, commonly referred to as "flowback," is often collected in open air evaporation pools, where water and volatile (likely to evaporate) hydrocarbons including methane will evaporate away, reducing the volume of fluids that must be transported for treatment. Although the evaporation of water is necessary to reduce treatment costs, the loss of volatile hydrocarbons produces an environmental hazard and is also a loss of product for the drilling company, since treatment of the flowback often yields additional liquid and gas fuels. Since methanol is a much less volatile compound than methane, the enzymatic conversion of methane to methanol will both prevent the release of methane into the atmosphere and increase the liquid fuel yield from flowback treatment.

Learn more about flowback treatment and our visions for improving it

Elimination System

In order to remove the methane dissolved in the flowback, our team wants to engineer a strain of bacteria that can survive in temporary flowback, storage tanks and convert the methane to methanol before the flowback is depressurized and transferred to the open air evaporation pool. Our first step in this project is to engineer E.coli that expresses soluble methane monooxygenase (sMMO), an enzyme capable of converting methane to methanol.

This protein is found exclusively in methanotrophs, a class of bacteria which are found in energy poor environments with high methane concentrations, from which they obtain their energy. The sMMO enzyme is composed of the proteins A, B, and C. Protein A is the hydroxylase which replaces one hydrogen on a methane with one hydroxyl group; it is composed of 2 symmetric dimers of subunits alpha, beta, and gamma. Protein C oxidizes NADH to acquire 2 electrons, which are then transferred by protein B to protein A for use in the methane oxidation pathway.

Previous attempts to clone sMMO into E. coli by other labs have successfully expressed proteins B and C, however, protein A while expressed, was non-functional, likely due to improper folding of the protein.

Process

The genes necessary to clone sMMO into E.coli are mmoX, mmoY, mmoZ, mmoB, and mmoC. The genes mmoX, mmoY, and mmoZ express protein A while mmoB expresses protein B and mmoC expresses protein C. The sequences of these genes were found in literature and optimized for cloning in E.coli. The optimized sequences could then be ordered and shipped to us for our use. After the genes are obtained in gene block format, they will be assembled using PCR, digestion, and ligation. Once assembled, they will be ligated into our varying strength expression vectors.

Project Status

The Biobricked sMMO genes are currently having RBS and promoter pairs of varying strengths inserted into the plasmids, with plans to characterize their different expression levels via 2D-SDS PAGE.

Currently, RBS's are being inserted into the sMMOX and sMMOY biobricks, while all four RBS and promoter pair crosses have already been created using sMMOZ and sMMOB. sMMOC has both high and low RBS inserted along with being ligated back into the standard vector and is in the process of promoter insertion.

Current Confirmed RBS, Promoter, sMMO Gene Biobricks
sMMO GeneHE RBS (promoterless)LE RBS (promoterless)HE Promoter/HE RBSHE Promoter/LE RBSLE Promoter/HE RBSLE Promoter/LE RBS
X
Y
Z √ √ √ √ √
B √ √ √ √ √
C √ √

Future

In the long run, our project’s end goal is to create a filtration system that directly treats waste water at fracking sites before the methane is allowed to evaporate out of the water. This filtration system will incorporate E.coli biofilm with our engineered sMMO that will convert methane into less volatile methanol as the waste water passes through to the REC enclosure. The methanol then can be used for other purposes, such as fuel. Our system will reduce the amount of methane contamination in the water and atmosphere, cut current treatment costs, and increase the renewability of fracking waste water.

References

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