Team:Michigan/Project
From 2009.igem.org
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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 Bba_K270003, 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 Bba_K270001 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. | 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 Bba_K270003, 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 Bba_K270001 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. | ||
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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 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. | ||
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In this design, a repression mechanism is downstream of the Pu promoter, and this this represses the production of holin and endolysin proteins. A constitutive promoter is placed in the front of the antiholin gene so antiholin is constantly produced. When toluene is present, it activates the Pu promoter which results in the repression of the promoter expressing holin and endolysin. As a result, the cell survives. However, in the absence of toluene, the repressor protein is not produced, so holin and endolysin levels rise to the point that the cells are lysed. | In this design, a repression mechanism is downstream of the Pu promoter, and this this represses the production of holin and endolysin proteins. A constitutive promoter is placed in the front of the antiholin gene so antiholin is constantly produced. When toluene is present, it activates the Pu promoter which results in the repression of the promoter expressing holin and endolysin. As a result, the cell survives. However, in the absence of toluene, the repressor protein is not produced, so holin and endolysin levels rise to the point that the cells are lysed. | ||
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The motivation for this design is as follows. The Berkeley 2008 design used an inducible promoter in front of the holin and endolysin; this inducible promoter then would trigger the onset of the killing mechanism. In our design, we wanted to use Pu to make the killing mechanism responsive to toluene concentration. However, we needed to set this up such that Pu would prevent cell death. Due to this requirement, we designed an inverter that is comprised of the repression module. This way, we did not have to modify the Berkeley part, which is characterized as working. | The motivation for this design is as follows. The Berkeley 2008 design used an inducible promoter in front of the holin and endolysin; this inducible promoter then would trigger the onset of the killing mechanism. In our design, we wanted to use Pu to make the killing mechanism responsive to toluene concentration. However, we needed to set this up such that Pu would prevent cell death. Due to this requirement, we designed an inverter that is comprised of the repression module. This way, we did not have to modify the Berkeley part, which is characterized as working. | ||
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It should be noted that this design requires the cells to be stored in media containing substrates that would activate the Pu promoter (e.g. toluene, o-xylene, methyl-benzene)in order to transport the cells to the actual contaminated site. This is because the cells would die in the absence of such substrates. | It should be noted that this design requires the cells to be stored in media containing substrates that would activate the Pu promoter (e.g. toluene, o-xylene, methyl-benzene)in order to transport the cells to the actual contaminated site. This is because the cells would die in the absence of such substrates. |
Revision as of 21:12, 21 October 2009
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