Demonstrate

Gold Medal Criterion #4

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 was filled with 900ul of 10mM Tris, pH 7.2, with 100uM TPA. 100um of enzyme mix supernatant was added to 4 wells, 100um of enzyme mix resuspended pellet was added to another 4, and water was added to 4 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.

12 well plate 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, since earlier tests suggested that the presence of cell lysate in the TPH enzyme mix may have impacted the growth of E. coli and driven up error. 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.

We are currently testing this protocol on PET samples that have been degraded by PETase, but we may run out of time before the wiki freeze. If PETase results are not here, we may have them at the jamboree!