Team:UC Davis/Data/Cutinase Activity

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The results of our first p-nitrophenyl butyrate (PNPB) assay shows that LC-cutinase appears to behave as an esterase, increasing the absorbance at 405 nm over time. We set up the run with both cutinase regulated by a constitutive promoter (BBa_J23101) and the inducible pBad promoter (BBa_K206000) in the MG1655 strain of <i>E. coli</i>. We also included a negative control of BBa_J04450 in MG1655 and another negative control of the PNPB buffer with LB instead of cells. The plot below displays the absorbance at 405 nm of each sample with the OD of the PNPB buffer control subtracted.
The results of our first p-nitrophenyl butyrate (PNPB) assay shows that LC-cutinase appears to behave as an esterase, increasing the absorbance at 405 nm over time. We set up the run with both cutinase regulated by a constitutive promoter (BBa_J23101) and the inducible pBad promoter (BBa_K206000) in the MG1655 strain of <i>E. coli</i>. We also included a negative control of BBa_J04450 in MG1655 and another negative control of the PNPB buffer with LB instead of cells. The plot below displays the absorbance at 405 nm of each sample with the OD of the PNPB buffer control subtracted.
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<center><img src="https://static.igem.org/mediawiki/2012/a/aa/UC_Davis_First_pNPB_Run.png"></center>
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Revision as of 01:15, 4 October 2012

Team:UC Davis - 2012.igem.org

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Cutinase Activity

As of now, through our p-nitrophenyl butyrate assays we have gathered enough data to determine that our LC-Cutinase part (BBa_K936000) exhibits its intended function as an esterase. However, it has been much more difficult to characterize the part than expected as we have been unable to purify the protein successfully.

The results of our first p-nitrophenyl butyrate (PNPB) assay shows that LC-cutinase appears to behave as an esterase, increasing the absorbance at 405 nm over time. We set up the run with both cutinase regulated by a constitutive promoter (BBa_J23101) and the inducible pBad promoter (BBa_K206000) in the MG1655 strain of E. coli. We also included a negative control of BBa_J04450 in MG1655 and another negative control of the PNPB buffer with LB instead of cells. The plot below displays the absorbance at 405 nm of each sample with the OD of the PNPB buffer control subtracted.


We attempted to redo the previous run while measuring OD 600 as to find the OD 405 per cell of each sample.


References

1. Silva C, et al. 2011. Engineered Thermobifida fusca cutinase with increased activity on polyester substrates. Biotechnol. J. 6:1230–1239.
2. S. Sulaiman, S. Yamato, E. Kanaya, J. Kim, Y. Koga, K. Takano, S. Kanaya. "Isolation of a Novel Cutinase Homolog with Polyethylene Terephthalate-Degrading Activity from Leaf-Branch Compost by Using a Metagenomic Approach." Applied and Environment Microbiology, vol. 78 no. 5, pp. 1556-1562, March 2012.
3. Ö. Faiz et al. Determination and characterization of thermostable esterolytic activity from a novel thermophilic bacterium Anoxybacillus gonensis J. Biochem. Mol. Biol., 40 (2007), pp. 588–594

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