Through our p-nitrophenyl butyrate assays we have gathered enough data to determine that our LC-Cutinase part (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K936000">BBa_K936000</a>) most likely 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. This has made many of the assays that we have conducted more difficult as most of the publications we followed used purified enzyme. A detailed description of these assays can be found on the Module Engineering Project page.

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.

We attempted to redo the previous run while measuring OD 600 as to find the OD 405 per cell of each sample. Below are the results for enzyme activity of cutinase expressed by a constitutive promoter (BBa_J23101), the inducible pBad promoter (BBa_K206000), and a negative control (BBa_J04450 in pSB1A2).