Team:Clemson/Project

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== '''Overall project''' ==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 ''che''Z 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.
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    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.
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== Project Details==
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      <p align="justify"><font size="5"><strong> <u>Abstract</u></strong></font></p><br>
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Polychlorinated biphenyls (PCBs) are widespread, cancer-causing pollutants left-over mainly from manufacture of capacitors and electric motors. There are over 200 possible PCBs, derivatives of biphenyl, which share the same biodegradation pathways in bacteria. Our team is using a genetic engineering approach to produce a small consortium of E. coli that should efficiently degrade biphenyl, and it is hoped that this same system can be adapted for the bioremediation of PCBs. Natural bioremediation by native bacterial communities is exceedingly slow due to the recalcitrant nature of PCBs and their hydrophobic properties which reduce the bioavailability to potential catabolizers. We are taking a three-pronged approach in an attempt to increase the efficiency of biphenyl bioremediation—attraction of biphenyl-degrading E. coli by other guiding bacteria, overexpression of the biphenyl catabolic enzymes, and production of a biosurfactant to increase the solubility of biphenyl. Together, this system should significantly increase the rate of biphenyl degradation.<br><br><br>
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      <p align="justify"><font size="5"><strong> <u>Motivation</u></strong></font></p><br>
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;From 1955 to 1987 in Pickens, South Carolina (the neighboring town to Clemson, SC) Sangamo-Weston Inc. had multiple factories, where they built capacitors. The dielectric fluid used in these capacitors contained Polychlorinated Biphenyls (PCBs). Due to improper land-burial of off-specification capacitors and wastewater treatment sludges that were built both on-site and off-site, PCBs contaminated the water supply of Lake Hartwell and its tributaries. Since PCBs have low water solubility, they remain mostly in the organic sediments and as particulate matter. Furthermore, PCB sediment concentrations are within the range of injury for benthic macroinvertabrates and exceed healthy levels in the fish of Lake Hartwell – an 87.5 square mile recreational lake that serves both South Carolina and Georgia areas.      </strong></p>
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  <p>&nbsp;</p>
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<br>
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  <p align="justify"><font size="5"><strong> <u>Introduction</u></strong></font></p><br>
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Overall, our project goal is to create a three-component bacterial system: signaling, biosurfactant, and PCB degradation. First is an expansion of the UT-Tokyo 2011 Team’s bioremediation specific system, consisting of guider and worker bacteria. The system (currently activated by UV light) causes the guider bacteria to produce Aspartate A, which is converted to L-Asp, a natural chemoattractant for E. coli. Thus, both guiders and workers will respond when the bacteria come into contact with the substrate. However, the reaction producing L-Asp is reversible and would cause the bacteria to no longer stay with the substrate. In order to circumvent this, UT-Tokyo created a system with a chromosomal cheZ knockout E. coli stain and initiated substrate-dependent cheZ expression from a plasmid. Thus, in the presence of the substrate, the lambda phage repressor cI is produced, binding to the cI-repressed promoter, inhibiting cheZ expression, and halting bacterial movement. In order for this system to work for our purposes, we are to replace the UV light promoter with a PCB specific promoter. Ideally we would like to implement this system in magnetotactic bacteria since PCBs are insoluble particles that remain in the sediments of Lake Hartwell, we will need the guiders to dive to the bottom. The workers in this system will be comprised of two genetically different bacteria: one programmed to produce a biosurfactant and the other to degrade the PCBs to less harmful byproducts.
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<br><br>
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            <img src="https://dl.dropbox.com/u/42151864/iGEM/Overview.png" frameborder="0"><br><p align="center"> <font size="2"><strong>Overview of Project Approach</strong></font></p>
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    <td><a href="http://www.clemson.edu/academics/programs/creative-inquiry/"><img src="http://f.cl.ly/items/3m1P441D0o343M1Y1u0E/cuci.PNG"  width="130" height="70" align="left"/></a></td>
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=== Part 2 ===
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=== The Experiments ===
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=== Part 3 ===
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== Results ==
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Latest revision as of 20:53, 19 April 2013

CU iGEM



Abstract


     Polychlorinated biphenyls (PCBs) are widespread, cancer-causing pollutants left-over mainly from manufacture of capacitors and electric motors. There are over 200 possible PCBs, derivatives of biphenyl, which share the same biodegradation pathways in bacteria. Our team is using a genetic engineering approach to produce a small consortium of E. coli that should efficiently degrade biphenyl, and it is hoped that this same system can be adapted for the bioremediation of PCBs. Natural bioremediation by native bacterial communities is exceedingly slow due to the recalcitrant nature of PCBs and their hydrophobic properties which reduce the bioavailability to potential catabolizers. We are taking a three-pronged approach in an attempt to increase the efficiency of biphenyl bioremediation—attraction of biphenyl-degrading E. coli by other guiding bacteria, overexpression of the biphenyl catabolic enzymes, and production of a biosurfactant to increase the solubility of biphenyl. Together, this system should significantly increase the rate of biphenyl degradation.


Motivation


     From 1955 to 1987 in Pickens, South Carolina (the neighboring town to Clemson, SC) Sangamo-Weston Inc. had multiple factories, where they built capacitors. The dielectric fluid used in these capacitors contained Polychlorinated Biphenyls (PCBs). Due to improper land-burial of off-specification capacitors and wastewater treatment sludges that were built both on-site and off-site, PCBs contaminated the water supply of Lake Hartwell and its tributaries. Since PCBs have low water solubility, they remain mostly in the organic sediments and as particulate matter. Furthermore, PCB sediment concentrations are within the range of injury for benthic macroinvertabrates and exceed healthy levels in the fish of Lake Hartwell – an 87.5 square mile recreational lake that serves both South Carolina and Georgia areas.

 


Introduction


     Overall, our project goal is to create a three-component bacterial system: signaling, biosurfactant, and PCB degradation. First is an expansion of the UT-Tokyo 2011 Team’s bioremediation specific system, consisting of guider and worker bacteria. The system (currently activated by UV light) causes the guider bacteria to produce Aspartate A, which is converted to L-Asp, a natural chemoattractant for E. coli. Thus, both guiders and workers will respond when the bacteria come into contact with the substrate. However, the reaction producing L-Asp is reversible and would cause the bacteria to no longer stay with the substrate. In order to circumvent this, UT-Tokyo created a system with a chromosomal cheZ knockout E. coli stain and initiated substrate-dependent cheZ expression from a plasmid. Thus, in the presence of the substrate, the lambda phage repressor cI is produced, binding to the cI-repressed promoter, inhibiting cheZ expression, and halting bacterial movement. In order for this system to work for our purposes, we are to replace the UV light promoter with a PCB specific promoter. Ideally we would like to implement this system in magnetotactic bacteria since PCBs are insoluble particles that remain in the sediments of Lake Hartwell, we will need the guiders to dive to the bottom. The workers in this system will be comprised of two genetically different bacteria: one programmed to produce a biosurfactant and the other to degrade the PCBs to less harmful byproducts.


Overview of Project Approach

 

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