Team:GeorgiaTech/Project

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.

The genes necessary to clone sMMO into E.coli are MMOX, MMOY, MMOZ, MMOB, and MMOC. The genes MMOX, MMOY, and MMOZ express the sub units of protein A while MMOB and MMOC express proteins B and C respectively. The sequences of these genes were found in literature and optimized for cloning in E.coli.

In comparison to previous attempts to express sMMO in E.coli, this project is novel in it's use of synthetic, E.coli codon optimized sMMO genes and it's use of a wide array of varying strength expression vectors. In order to effectively regulate the translation and transcription rates for the genes a number of insertion primers have been designed to turn our biobrick parts into expression vectors by inserting Ribosomal binding sites (RBS) and promoters into the PSB-1C3 plasmid (effectively eliminating months of unsuccessful ligations with small regulatory sequences). It is hypothesized that finding the right expression level for each sMMO gene relative to each other and the cell as a whole could create an "optimum mix" of sMMO proteins that minimizes protein misfolding and inclusion body formation and maximizes functional sMMO enzyme production.

In order to characterize the function of the sMMO complex, the proposed methods included a 2D SDS PAGE as well as conducting a colorimetric plate assay for sMMO activity. The 2D SDS PAGE would show the production of sMMO proteins in the cell (functional or not) when compared against a control E.coli strain. On the other hand, the colorimetric assay would be able to assess the function of the entire sMMO protein complex. The colorimetric assay is a straight-forward method for screening methanotrophic colonies that express sMMO activity on solid media. The sMMO activity would result in the development of a colored complex which occurs when the sMMO reacts with naphthalene, and o-dianisidine. We would expect the methanotrophic colonies that express the sMMO to turn deep purple when exposed to these compounds.

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, and promoters are being inserted into the sMMOC Biobrick. All four RBS and promoter pair crosses have already been created using sMMOZ and sMMOB.

Current Confirmed RBS, Promoter, sMMO Gene Biobricks

sMMO Gene	X	Y	Z	B	C
HE RBS (promoterless)			√	√	√
LE RBS (promoterless)			√	√	√
HE Promoter/HE RBS			√	√
HE Promoter/LE RBS			√	√
LE Promoter/HE RBS			√	√
LE Promoter/LE RBS			√	√

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.

Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum.