Team:UMaryland/Measurement
Measurement
PET degradation has been and is a popular iGEM project (see our review!). However, measurement of this degradation remains a challenge for three major reasons. First, the fastest PET degrading enzyme currently on the registry, PETase, is highly specific for PET. This means that despite its superior degrading ability, it will actually produce weaker results when tested with a PNPB esterase assay than less powerful enzymes like LC cutinase (joo et. al.). This assay has been very popular with iGEM teams, but can not be used to determine which of two enzymes is more effective at PET degradation.
Figure from Yoshida et. al. Despite being much more effective at degradation of PET, PETase produces minimal results in a PNPB absorbance assay.
Second, enzymatic degradation of PET is glacially slow. No iGEM team has been able to confirm PET degradation on the basis of visual inspection or weight. The only way this degradation has been successfully detected is with powerful instruments such as SEM, mass spec, HPLC, and cell-free expression. Teams without access to these expensive resources have no way to obtain results yet.
Very pricey white benchtop machines that detect PET degradation (SEM, LCMS, HPLC)
The third challenge lies in the nature of PET degradation byproducts. These include MHET, ethylene Glycol, and TPA. Ethylene glycol is metabolized by E. coli, and teams have not been able to to determine enzymatic efficiency based on cell growth from ethylene glycol production. MHET is converted to TPA and ethylene glycol by MHETase, which makes TPA the most relevant target when quantifying degradation. However, TPA has poor solubility in water of about 100uM. Thus, any method to detect TPA from PET degradation must be extremely sensitive.
A popular figure on PETase byproducts and the role of MHETase
Our project employs a fluorescent biosensor from Los Alamos, PcaU, that is the product of directed evolution for high sensitivity to a downstream byproduct of TPA metabolism: Protocatechuic acid (PCA). We have shown that this sensor differentiates single micromolar PCA concentrations and can be used to detect TPA. Teams will be able to quantify PET degradation through bacterial fluorescence without need for expensive instruments. The quantitative cell-based sensor has potential use in the directed evolution of PETase as well. Notably, many of the latest articles of PETase mention that the enzyme holds promise for substantial improvement, making the promise of such an approach quite exciting. Details on the PcaU biosensor are available in its registry page.
The lower boundary of PCAU detection range, n=8. Such sensitivity is necessary if we hope to detect and quantify TPA production from PET degradation
Inspiration
You can look at what other teams did to get some inspiration!
Here are a few examples: