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Future Work

Where We Stand

S.A.F.E.B.E.T. is more than a proof of concept project. We designed a unique modular biosensing platform and built a functional field-deployable device, but the platform is not perfect and the device can be improved. The biosensing platform is modular in that inducible promoters can be inserted into our genetic construct relatively easily – any promoter that is upregulated in response to a toxin can be inserted into our system just like a BioBrick. We implemented the biosensing platform by actually designing and building a field-deployable device. The device is designed to operate independently for 6 months in the field but can be controlled remotely via a mobile/internet application. Taken together, our biosensing platform and device is (nearly) a finished product. Soon we can deploy an operational biosensor that will provide useful data on arsenic and naphthalene levels, but our team aspires for more than “good enough.” S.A.F.E.B.E.T. currently relies on antibiotic resistance as a selective pressure to maintain plasmids in Shewanella oneidensis. We also require a second ~11kb plasmid in addition to the plasmid with our inducible reporter system in order to detect naphthalene. Our device is designed to float at the surface of a river in order to detect toxins and be easier to access if maintenance is required. We are cognizant of how to develop S.A.F.E.B.E.T. into a one-of-a-kind tool to monitor toxins in remote waterways and large-scale watershed networks.

Auxotrophy as Means of Selection

Antibiotic resistance genes are powerful tools for molecular biologists, but potentially dangerous if the wrong organisms obtain the resistance genes. One alternative to using antibiotic resistance as a selective pressure to maintain plasmids in bacteria is to engineer bacteria to be auxotrophic for a critical cell metabolite, then re-introduce the genes(s) on the plasmid. One downside to using plasmids is that they are energetically costly to replicate and express genes from and can slow down the overall growth and metabolism and thus electric current output of S. oneidensis. We devoted a lot of time and thought into developing our unique strains of S. oneidensis - another benefit of making our strains auxotrophic is that they will not survive in the wild without our device providing dietary supplements.

Chromosomal Integration

A small plasmid with few expressed genes may not affect the current output of S. oneidensis to a significant degree, but a large plasmid with many expressed genes (such as our secondary naphthalene degradation plasmid) significantly impairs the growth and metabolism of S. oneidensis. Integrating the naphthalene degradation operon into the chromosome of S. oneidensis may help partially alleviate the energy cost of replicating several copies of a huge plasmid. In addition to alleviating the stress caused by expressing a giant operon, integrating our genetic parts into the chromosome eliminates the need to design a selective pressure for S. oneidensis to maintain extrachromosomal DNA.

Proteolysis Tag

Proteins can be tagged for degradation by proteases with a proteolysis tag. By fusing such a tag to MtrB, we can tell the cell to degrade the protein at a higher rate, allowing us to decrease the steady state concentration of MtrB at all levels of analyte. If we are able to tune the degradation of MtrB such that its concentration at uninduced levels is not sufficient to complex with available MtrA and MtrC, the basal current production that our engineered strains produce would be decreased. Consequently, the dynamic range of our biosensor would be increased, since higher levels of analyte would be needed to generate the promoter activity requisite to produce MtrB in sufficient quantity to fully localize all MtrA and MtrC—i.e., current production would saturate at higher levels of analyte.