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| - | <h1>Cutinase Activity</h1> | + | <h1>Cutinase Expression and Activity</h1> |
| | <article> | | <article> |
| - | Through our p-nitrophenyl butyrate (pNPB) 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. | + | Through our testing of LC-Cutinase expressing and activity we have determined that (1) pelB is working to transport the catalyst to the extracellular matrix and that (2) the expressed cutinase exhibits esterase activity. |
| | <br><br> | | <br><br> |
| - | 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 <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. | + | <b>Expression and Secretion</b><br> |
| - | | + | We conducted westerns while probing for the 6x-his tag to determine whether or not pelB was working as a secretion mechanism for LC-Cutinase. The western below shows supernatant media samples of a culture expressing the gene. A single band is clearly seen around 30 kDa, which corresponds to the expected size of LC-Cutinase. This tells us that pelB is indeed working to secrete the enzyme. |
| - | | + | <br><center><img src="https://static.igem.org/mediawiki/2012/d/d9/UCDavisParts2.png"></center> |
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| - | <br><br><center ><a href="https://static.igem.org/mediawiki/2012/a/aa/UC_Davis_First_pNPB_Run.png" class="lightbox"><img src="https://static.igem.org/mediawiki/2012/ c/ c7/ UCD_Data_12. jpg " > </a> </center> <br> | + | When looking at this expression over time, however, we see an unexpected trend in enzyme production and secretion. The below figure shows the intensity of bands on the western (integrated using ImageJ) over different time points after induction. While much protein is found in the media after 1 hour, the level seems to then decrease. We are currently conducting further characterization of the expression and secretion of LC-Cutinase in hope of better quantifying how it is expressed. |
| - | | + | <br><center><img src="https://static.igem.org/mediawiki/2012/c/c4/UCDavisParts1.png"></center> |
| - | 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.
| + | <br><br> |
| - | 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.
| + | <b>Cutinase Activity</b><br> |
| | + | Through our p-nitrophenyl butyrate (pNPB) 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>) exhibits its intended function as an esterase. The results below were acquired through assays conducted with cell culture samples. We used cell cultures in these runs because in actual application we would like to incubate cells directly with PET and because given the results of our last experiments, we are confident that pelB is working to secrete the enzyme. A more detailed description of these assays can be found on the Module Engineering Project page. |
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| | + | The first of these runs was conducted with cutinase regulated by both a constitutive promoter (BBa_J23101) and the inducible pBad promoter (BBa_K206000) in the MG1655 strain of <i>E. coli</i>. We included a negative control of BBa_J04450 in MG1655 and another negative control of the pNPB buffer with LB instead of cells to get an idea of any background esterase activity. The figure below displays the absorbance at 405 nm of each sample after 8 hours with the OD of the pNPB buffer control subtracted. The catalytic activity of cells expressing LC-Cutinase is clearly higher than that of the background. |
| | + | <br><center><img src="https://static.igem.org/mediawiki/2012/0/0b/UCDavisParts3.png"></center> |
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| - | <a href="https://static.igem.org/mediawiki/2012/e/e2/UCD_Data_large_13.jpg" class="lightbox"><img src="https://static.igem.org/mediawiki/2012/0/0e/UCD_Data_13-1.jpg " align="left"> </a>
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| - | <a href="https://static. igem. org/mediawiki/2012/b/bb/UCD_Data_large_14.jpg" class="lightbox"><img src=" | + | 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) and a negative control (BBa_J04450 in pSB1A2). |
| - | https://static.igem.org/mediawiki/2012/c/cb/UCD_Data_14.jpg " align="right"> </a> | + | These results show that cutinase has higher catalytic activity than that of the background, but not by the same relative amount as we saw in the previous run. |
| - | | + | <br><center><img src="https://static.igem.org/mediawiki/2012/ 6/ 63/ UCDavisParts4. png"></ center> |
| - | <br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>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. | + | <br><br> |
| - | <br><br><center>
| + | When repeating this run a third time, we see a similar trend. Cutinase activity is higher than the background, but at a different relative amount. |
| - | <a href="https://static.igem.org/mediawiki/2012/f/f3/UCD_Data_large_15.jpg" class="lightbox"><img src="https://static.igem.org/mediawiki/2012/ c/ c8/ UCD_Data_15. jpg" ></a></center> | + | <br><center><img src="https://static.igem.org/mediawiki/2012/2/28/UCDavisParts5.png"></center> |
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| - | <br> | + | These results confirm that LC-Cutinase does demonstrate esterase activity but again suggest that the expression is variable and that further characterization is needed. |
| - | 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.
| + | <br><br> |
| | + | To try to test cutinase activity in a more controlled environment, we tried to purify the enzyme by way of its 6x-His tag. Unfortunately, after a couple of attempts, we were not successful. One potential explanation to why we are having such a difficult time purifying the enzyme is it could be possible that the C-terminal 6x-His tag is inaccessible to our purification kits. If this is the case, we could investigate the possibility of adding a N-terminus tag, and see if we would be able to purify the protein through that alternate method. |
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