Team:Cornell/testing/project/wetlab/5

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<h3>Where We Stand</h3>
<h3>Where We Stand</h3>
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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 naphthalane. 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.
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S.A.F.E.B.E.T.  is more than a proof of concept project.  We have designed a unique, modular, biosensing platform and have built a functional field-deployable prototype. Furthermore, by engineering our constructs with cut-sites flanking the sensing module, we have created a modular platform that can be readily utilized for the detection of different analytes. Despite this versatility, several steps can be taken to further improve our device and platform.
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<h3>Auxotrophy as a means of plasmid maintenance and preventing release of our GEMs</h3>
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Antibiotic resistance genes are powerful tools for molecular biologists, but potentially dangerous due to horizontal gene transfer.  One alternative method of selective pressure is to engineer bacteria auxotrophic to a critical cell metabolite (e.g. phenylalanine, uracil, etc.). By re-introducing the necessary gene(s) on a plasmid, our constructs could be sustainably maintained without the fear of imparting antibiotic resistance to environmental bacteria strains. Additionally, our auxotrophic strains would be unable to survive outside of the device.
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<h3>Auxotrophy as Means of Selection </h3>
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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.
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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.
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<h3>Chromosomal Integration </h3>
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<h3>Chromosomal integration as a means to alleviate energetic cost of maintaining plasmids</h3>
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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.
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The maintenance of plasmids is both a metabolic and energy stress on the cell. This is particularly true if many genes are encoded on the plasmid. Due to the sheer size of our naphthalene degradation construct, maintenance runs the risk of  impairing both growth and replication of our engineered <em>Shewanella</em>. This has the potential to  interfere with the sensitivity and accuracy of our biosensing platform. By integrating the naphthalene degradation operon into the chromosome of our strains, we would aim to alleviate the energy cost of maintaining our construct. Additionally, chromosomal integration would lower the copy number of our constructs, preventing the current from saturating at basal levels and providing a larger dynamic range.
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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.
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<h3>Proteolysis tag as means to lessen the effects of leaky expression</h3>
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<h3> Proteolysis Tag </h3>
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By fusing a proteolysis tag to MtrB, MtrB would be degraded by proteasomes at a higher rate. This would allow us to tune the degradation of MtrB such that protein concentrations at non-induced levels are insufficient for complexing with other components of the Mtr pathway and hence, at producing current. By decreasing the basal current levels of our strains, the dynamic range of our biosensing platform would be increased.
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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&#8212;<i>i.e.</i>, current production would saturate at higher levels of analyte.
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Latest revision as of 03:58, 4 October 2012

Future Work

Where We Stand

S.A.F.E.B.E.T. is more than a proof of concept project. We have designed a unique, modular, biosensing platform and have built a functional field-deployable prototype. Furthermore, by engineering our constructs with cut-sites flanking the sensing module, we have created a modular platform that can be readily utilized for the detection of different analytes. Despite this versatility, several steps can be taken to further improve our device and platform.

Auxotrophy as a means of plasmid maintenance and preventing release of our GEMs

Antibiotic resistance genes are powerful tools for molecular biologists, but potentially dangerous due to horizontal gene transfer. One alternative method of selective pressure is to engineer bacteria auxotrophic to a critical cell metabolite (e.g. phenylalanine, uracil, etc.). By re-introducing the necessary gene(s) on a plasmid, our constructs could be sustainably maintained without the fear of imparting antibiotic resistance to environmental bacteria strains. Additionally, our auxotrophic strains would be unable to survive outside of the device.

Chromosomal integration as a means to alleviate energetic cost of maintaining plasmids

The maintenance of plasmids is both a metabolic and energy stress on the cell. This is particularly true if many genes are encoded on the plasmid. Due to the sheer size of our naphthalene degradation construct, maintenance runs the risk of impairing both growth and replication of our engineered Shewanella. This has the potential to interfere with the sensitivity and accuracy of our biosensing platform. By integrating the naphthalene degradation operon into the chromosome of our strains, we would aim to alleviate the energy cost of maintaining our construct. Additionally, chromosomal integration would lower the copy number of our constructs, preventing the current from saturating at basal levels and providing a larger dynamic range.

Proteolysis tag as means to lessen the effects of leaky expression

By fusing a proteolysis tag to MtrB, MtrB would be degraded by proteasomes at a higher rate. This would allow us to tune the degradation of MtrB such that protein concentrations at non-induced levels are insufficient for complexing with other components of the Mtr pathway and hence, at producing current. By decreasing the basal current levels of our strains, the dynamic range of our biosensing platform would be increased.