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TPA ENZYME MIX

Enzymatically converting TPA to PCA for detection

Results

The enzymes needed to convert TPA to PCA are available in the registry from Darmstadt 2012. By the time PcaU was syntesized, we did not have enough time to use Darmstadt’s parts which required assembly. We contacted professor Eiji Masai, who generously provided us with the necessary plasmids to express the enzymes in E. coli. These are pETtpa23, pRSFtpa1 and pETtpb from Masai’s 2006 paper.

Figure from Masai et. al.
The terepthalic acid degradation pathway. It is an aerobic process that requires the presence of NADPH, although the NADPH is restored at the end of the proccess.

The plasmids were expressed in BL21 DE3 E. coli and protein was prepared using the protocol below.

We tested TPH enzyme functionality in various buffers to develop a final buffer, TPH buffer. TPH buffer is a buffer that must maintain tph enzyme function in the presence of PETase buffer. This is because the resuspended tph enzymes will be applied to supernatant from PETase degradation containing TPA. The tph enzymes will convert TPA to PCA for detection by PcaU. Therefore, these buffers must also not prevent growth of PcaU transformed E. coli cells. 10mM Tris buffer at pH 7.2 was chosen for PETase buffer and TPH buffer. PNPB tests from multiple teams including our own have shown that petase is functional in Tris, and our own growth tests revealed that although Tris may be somewhat toxic, PcaU transformed BL21 E. coli are able to grow in the presence of 10mM tris buffer. Tph buffer also contains some key components used in Masai’s work, including 0.1mM ferrous ammonium sulfate (required iron supply), 2mM L-Cysteine Hydrochloride, and 10% glycerol (for preservation). We also added 100um NADPH to this buffer to speed the reaction

PETase has worked in PBS, but tph enzymes may not due to NADPH being unstable in phosphate buffers. PBS also precipitated when ferrous ammonium sulfate was added to it, likely due to formation of insoluble ferrous ammonium phosphate. PBS is not a suitable buffer for TPH enzymes

PETase activity test, testing supernatant of PET plastic exposed to PETase lysate for one week. Error level is high due to the 24 well plate hitting the wall of the shaker, resulting in the plate needing to be read 2 hours after PcaU induction. The hit caused some media to leak from the sides of wells, which was partially corrected for by dividing fluorescence over absorbance. The results on this test are not conclusive, but they are promising. Knowing that 25uM TPA is distinguishable from a negative control after 6 hours with our approach, the fact that the average fluorescence for degraded PET is noticeably higher than our TPA positive control leads us to predict that the assay is capable of producing results.

It is unusual that PETase would produce such high fluorescence, however, because that would require a great amount more TPA that 25uM. In fact, it would require more than should be able to dissolve in water. This may have been a result of active PETase still being present in the supernatant when the TPH enzyme mix was added. Thus, as the TPH enzyme mix was converting TPA to PCA during the overnight incubation, PETase may have been producing more TPA from PET byproducts left over in the supernatant.

The test above 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, and if we manage to do so 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!