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The results of our first 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. 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). The plot on the right includes the results for the pBad expressed cutinase mutants described in the Protein Engineering section. These results show that some of the mutants may have higher activity but do not confirm the findings of the previous run suggesting that cutinase has a higher esterase activity than the negative control.
We ran the assay once again with only constitutively expressed cutinase (with BBa_J23101) and the negative control (BBa_J04450 in pSB1A2). This time, the results reasserted the initial finding that the expressed cutinase had a higher activity than the control.
From the first and last results, we can conclude that the LC-cutinase catalyst most likely behaves as expected. That is to say that it behaves as an esterase and breaks down the pNPB in the assay at a distinguishable rate. However, the middle results show inconsistencies that suggest that we should conduct more runs in the future. We are currently working to purify the cutinase enzyme which will allow us to redo these runs with a standardized enzyme concentration. This will allow for more repeatable and reliable results.
