Team:UMaryland/Demonstrate
Demonstrate
Detecting TPA with PcaU
Results
The PCA sensor plasmid we assembled contains a GFP gene whose expression is regulated by a promoter repressed by the PCA-induced repressor PcaU. When TPA is converted to PCA using TPH enzyme mix, the operon binds PCA and is de-repressed, leading to increased GFP fluorescense. Multiple tests on this sensor have demonstrated its capability of distinguishing single-micromolar differences in added PCA concentration when expressed in BL21(DE3) E. coli. The sensor was also found to be functional in DH5α cells, although it was not as sensitive. Further information and characterization of PcaU’s response to PCA is available on the part’s registry page.
Below, BL21(DE3) E. coli containing the PcaU-based PCA sensor were used to detect and confirm TPA degradation (TPA in turn being a product of PET degradation) from the TPH enzyme mix. The bar graph of Figure 1 below shows GFP fluorescence emission at 509 nm from the PcaU sensor cells exposed to the products of enzymatic conversion of TPA to PCA (40uM TPA in buffer with added TPH enzyme mix) vs. a control (40uM TPA in buffer).
Figure 1
This is a 12 well plate TPA activity assay, n = 4. Wells in a 12 well plate were filled with 900ul of 10 mM Tris, pH 7.2, with 100 uM TPA. 100 uM of enzyme mix supernatant was added to test wells, and water was added to control wells. The plate was incubated at 30 °C in aerobic conditions overnight to enzymatically convert TPA to PCA. In a separate flask, PcaU sensor cells were grown to OD600=0.6, and 1 mL of culture was added to each well, diluting the original TPA concentration to a final 45uM. Fluorescence was measured in a plate reader eight hours later, with 395nm excitation, 509nm emission. A significant difference was observed between supernatant and control.
This is a 12 well plate TPA activity assay, n = 4. Wells in a 12 well plate were filled with 900ul of 10 mM Tris, pH 7.2, with 100 uM TPA. 100 uM of enzyme mix supernatant was added to test wells, and water was added to control wells. The plate was incubated at 30 °C in aerobic conditions overnight to enzymatically convert TPA to PCA. In a separate flask, PcaU sensor cells were grown to OD600=0.6, and 1 mL of culture was added to each well, diluting the original TPA concentration to a final 45uM. Fluorescence was measured in a plate reader eight hours later, with 395nm excitation, 509nm emission. A significant difference was observed between supernatant and control.
The Darmstadt team decided not to pursue testing of the TPH enzymes in 2012, so this is the first confirmation of these enzymes’ functionality in E. coli lysate by an iGEM team.
The sensor’s response to changes in TPA concentration was also characterized. Below, we show that despite substantial background from leaky expression, the response to TPA is clearly detectable, and possibly saturated, at 22.5 uM TPA.
Figure 2
TPA detection assay, n=4. Each well was filled with 900ul of 10mM Tris, pH 7.2, with 4 wells containing 100 um TPA, 4 containing 50 um TPA, and 4 containing no TPA. 100ul of TPH enzyme mix was added to each well and plate was incubated at 30C aerobically overnight to enzymatically convert TPA to PCA. PcaU BL21 cells were grown to OD600=0.6, and 1 mL culture was added to each well, diluting samples to 45uM TPA and 22.5uM TPA final concentrations. Fluorescence was measured 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.
TPA detection assay, n=4. Each well was filled with 900ul of 10mM Tris, pH 7.2, with 4 wells containing 100 um TPA, 4 containing 50 um TPA, and 4 containing no TPA. 100ul of TPH enzyme mix was added to each well and plate was incubated at 30C aerobically overnight to enzymatically convert TPA to PCA. PcaU BL21 cells were grown to OD600=0.6, and 1 mL culture was added to each well, diluting samples to 45uM TPA and 22.5uM TPA final concentrations. Fluorescence was measured 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 thus demonstrated efficacy for detecting the presence of a PET degradation byproduct, TPA. If the sensor is to be used for directed evolution, it must be capable of responding robustly to a range of TPA concentrations. Improving assay and sensor sensitivity to achieve this result could be accomplished in future experiments. The first action we would pursue is His-tagging and purification of the TPH enzymes to remove any potential interfering factors present in cell lysate. A lab with more resources and time would also be able to express TPH enzymes and the PcaU sensor in the same cell. This would remove the necessity for cell lysis, and it would permit single cell analysis of TPH activity through flow cytometry.
An attempt was also made to detect TPA in a solution that contained PET treated with PETase, 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 based on the results from previous papers, which have obtained TPA concentrations of up to 100 uM via PETase degradation of PET, as can be seen in the medley of figures below. The figures also show that engineering of PETase itself is possible. You will see our results at the Jamboree!
Figure 3
PETase results from Joo et. al (Top), Yoshida et. al. (Left), and Beckham et. al (Right). PETase reactions generate TPA at concentrations similar to those detected here, and engineering of PETase can control enzymatic rates and product distributions.
PETase results from Joo et. al (Top), Yoshida et. al. (Left), and Beckham et. al (Right). PETase reactions generate TPA at concentrations similar to those detected here, and engineering of PETase can control enzymatic rates and product distributions.
In conclusion, there are many, many variables to account for in this system. However, with 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 in vivo fluorescent detection will be unleashed!
bottom figure sources:
Top: Joo, S. et. al. Structural insight into molecular mechanism of poly(ethylene terephthalate) degradation. Nature Communications 9 (2018)
Right: Beckham, G. et al. Characterization and engineering of a plastic-degrading aromatic polyesterase. PNAS 115 (2018).
Left: Yoshida, S. et al. A bacterium that degrades and assimilates poly(ethylene terephthalate). Science 351, 1196–1199 (2016).
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