Team:Michigan/Project
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- | The Toluene Terminator is a ''Pseudomonas'' device that aims to identify the toxic compound toluene in an environmental setting (e.g. a spill into soil from an underground petrol tank), move to and uptake it, metabolize it, and destroy itself when all of the toluene has been metabolized. This device will require four basic modules: toluene chemorecognition, chemotaxis, toluene metabolism, and a suicide mechanism. This year we | + | The Toluene Terminator is a ''Pseudomonas putida'' device that aims to identify the toxic compound toluene in an environmental setting (e.g. a spill into soil from an underground petrol tank), move to and uptake it, metabolize it, and destroy itself when all of the toluene has been metabolized. This device will require four basic modules: toluene chemorecognition, chemotaxis, toluene metabolism, and a suicide mechanism. This year we focused on toluene metabolism and the suicide mechanism. |
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- | The goal of this project is to work with the toluene degrading abilities that already exist in ''P. putida'' mt-2 (ATCC 33015) on the pWW0-TOL plasmid. The pathway on this plasmid is comprised of the upper pathway where toluene is metabolized into catechol and the lower | + | The goal of this project is to work with the toluene degrading abilities that already exist in ''P. putida'' mt-2 (ATCC 33015) on the pWW0-TOL plasmid. The pathway on this plasmid is comprised of the upper pathway, where toluene is metabolized into catechol, and the lower meta-pathway, where catechol is converted into acetaldehyde and pyruvate. The regulation of toluene degradation on the pWW0 plasmid is presented below. |
- | [[Image: | + | [[Image:Toluene_degradation_topology2.jpg|950px]] |
The image is adapted from Burlage, R. S., S. W. Hooper, and G. S. Sayler. 1989. The TOL (pWWO) catabolic plasmid. Appl. Environ. Microbiol. 55: 1323-1328. | The image is adapted from Burlage, R. S., S. W. Hooper, and G. S. Sayler. 1989. The TOL (pWWO) catabolic plasmid. Appl. Environ. Microbiol. 55: 1323-1328. | ||
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- | Our project is aimed at characterizing the promoters in the pWW0 plasmid to sense when the toluene degrading process is finished, signaling the cell to commit suicide. The promoter we chose to sense the presence of toluene is the Pu promoter. In order to characterize this promoter we created | + | Our project is aimed at characterizing the promoters in the pWW0 plasmid to sense when the toluene degrading process is finished, signaling the cell to commit suicide. The promoter we chose to sense the presence of toluene is the Pu promoter. In order to characterize this promoter we created [http://partsregistry.org/wiki/index.php?title=Part:BBa_K270003<font color=navy><B>Bba K270003</B></font>], a device that has the Pu promoter expressing GFP. To have this device function in strains other than ''P. putida'' mt-2 that already contains the XylR regulating protein, the part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K270001<font color=navy><B>Bba K270001</B></font>] was created from the Pr XylR portion of the pWW0 plasmid to regulate the Pu promoter. By combining the functions of these two parts, the intensity of GFP can be used to measure the promoter activity when induced with non-lethal level of toluene. |
- | Currently, we have made the Pu GFP and Pr XylR parts. In the future, we plan on creating one device composed of these two parts and testing the Promoter activity in both ''E. coli'' and ''P. putida''. With this data we will have a better idea of how to link the toluene sensing ability of the Pu promoter with suicide mechanism. | + | Currently, we have made the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K270003<font color=navy><B>Pu GFP</B></font>] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K270001<font color=navy><B>Pr XylR</B></font>] parts. In the future, we plan on creating one device composed of these two parts and testing the Promoter activity in both ''E. coli'' and ''P. putida''. With this data we will have a better idea of how to link the toluene sensing ability of the Pu promoter with suicide mechanism. |
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[[Image:Kill_switch_topology_for_modeling.jpg|950px]] | [[Image:Kill_switch_topology_for_modeling.jpg|950px]] | ||
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+ | ==<B><font size=5>Chemotaxis</font></B>== | ||
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+ | We wanted to link chemotaxis to our project to have the the bacteria seek out toluene in contaminated environments. We planned on engineering ''P. putida'' to swim up toluene gradients. We did a few initial studies to characterize the natural motility of ''P. putida'' and our results showed that ''P. putida'' has chemotaxis abilities. After this first initial study we focused on the other parts of the project. | ||
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Latest revision as of 01:21, 22 October 2009
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