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.
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