Team:Clemson

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In an effort to fight pollution through the process of bioremediation, our team proposes to develop bacteria that will be highly effective in degrading Polychlorinated Biphenyls (PCBs) with a long term goal of being able to help clean Lake Hartwell, a major recreational area for people of all ages in the upstate of South Carolina. To accomplish this, we have adapted and expanded the model constructed by the 2011 UT Tokyo team. The focus of their project was to engineer a division of bacteria that would increase the efficiency of bioremediation in any given environment. Their two-component system is comprised of a cell assembling and a cell arrest system. Within the parameters of the cell assembling system, their employed mechanism was to use chemoattraction to increase the density of bacteria at a desired location. To accomplish this, aspartate was used as a natural chemoattractant, and its production was induced by the presence of the substrate. The cell arrest system, a secondary extension of system one, is used to maintain a high density of bacteria once they have reached the desired location since it is likely that the concentration of bacteria will dissipate as the substrate is degraded. This system operates by “arresting” or prohibiting the rotation of the flagellum by regulation of the cheZ gene and thereby decreasing cell motility. Following a few key modifications, such as a change in promoters, this system will be adjusted to work perfectly for our project.

As part of the goal of this project to degrade PCB’s, we have found need for a biosurfactant. Such a compound would serve to emulsify the hydrophobic PCB’s and make them available to the degrading microbes. At first, it was thought that cloning the genes necessary for biosurfactant production into the swarming microbes would be too inefficient and that a natural biosurfactant-producing microbe would need to be part of the system. However further research showed that Pseudomonas aeruginosa produces rhamnolipids, a class of glycolipid often regarded as among the best of bacterial-produced surfactants. It was also determined that the genes necessary for this rhamnolipid production could potentially be isolated and cloned into Escherichia coli. In addition, several strains of P. aeruginosa were readily available, making it an obvious choice. The rhamnolipid genes of several strains were compared and found to be highly conservative. This has lead to the creation of primers for the PCR that would isolate and amplify these genes making them available for cloning into the swarming microbes of the PCB degrading system. For the actual PCB/ biphenyl degradation, we plan to clone genes from Spingobium yanoikuyae using PCR, which will then be activated after our microbial community has sensed biphenyl in the environment. So far, we are in the process of designing primers for the array of genes required for biphenyl degradation.