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PET NET: Improve

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Detecting TPA with PcaU

Results

PcaU was tested and shown to be capable of distinguishing single micromolar differences in pure PCA concentration when inside BL21 DE3 E. coli. The sensor was also found to be functional in 5a cells, although not as sensitive. Further information and characterization of PCAU’s sensitivity to PCA is available on the part’s registry page.

12 well plate TPA activity assay, n=4. Each well in a 12 well plate was filled with 900ul of 10mM Tris, pH 7.2, with 100uM TPA. 100um of enzyme mix supernatant was added to test wells, and water was added to control wells. Plate was incubated at 30C overnight to enzymatically convert TPA to PCA. PcaU BL21 cells were grown to OD600=0.6, and 1mL culture was added to each well, diluting original TPA concentration to a final 45uM. Fluorescence was taken in a plate reader eight hours later at 395nm excitation, 509nm emission. A significant difference was observed between supernatant and control. The pellet failed to produce a significant result.

TPA detection assay, n=4. Each well was filled with 900ul of 10mM Tris, pH 7.2, with 4 containing 100um TPA, 4 containing 50um TPA, and 4 containing no TPA. 100ul of tph enzyme mix was added to each well and plate was incubated at 30C overnight to enzymatically convert TPA to PCA. PcaU BL21 cells were grown to OD600=0.6, and 1mL culture was added to each well, diluting samples to 45uM TPA and 22.5uM TPA final concentrations. Fluorescence was taken in a plate reader six hours later at 395nm excitation, 509nm emission. A significant difference was observed between culture exposed to degraded TPA and the control. The difference between 22.5uM TPA and 45uM TPA was not significant.

This protocol has demonstrated efficacy for detecting the presence of a PET degradation byproduct, TPA. However, the magnitude of error results in an inability to distinguish between certain concentrations of product. If the sensor is to be used for directed evolution, it must be capable of doing this. Improving sensor resolution to achieve this result could be accomplished in a variety of manners. The first action we would pursue is HIS purification of the TPH enzymes to remove any potential interfering factors present in cell lysate. A lab with more resources and time would be able to express TPH enzymes and PCAU in the same cell. Not only would this drive down error from exposure to cell lysate, but it would permit single cell analysis of TPH activity through flow cytometry.

PETase results from Joo et. al (Top), Yoshida et. al. (Right), and Beckham et. al (left) exhibit up to 100uM TPA

A test was conducted to detect TPA in PETase treated solution containing PET, but the multi well plate fell off our shaker. The test will be repeated, but data will not be available in time for the wiki freeze. We are optimistic that we can conclusively detect PET degradation due to the results from previous papers, which have obtained TPA concentrations of up to 100uM via PETase degradation of PET, as can be seen in the medley of figures above. You will see our results at the Jamboree!

In conclusion, there are many, many variables to account for in this system. However, with enough continued testing and optimization we are confident it will be possible to quantify PET degradation using this system. Then, the potential of directed PETase evolution via cell fluorescence will be unleashed!