Team:UMaryland/TPAvPCA
Bacterial Cellulose
Does the addition of a cellulose binding domain accelerate the degradation of PET?
When we ran into the issue of detection of the degradation of PET, we found two potential sensor systems that we would be able to use, one which detected protocatechuate (PCA) and the other detecting terephthalate (TPA). To determine which sensor system was better for our needs, we used the MatLab SimBiology package to model the degradation of PET down to PCA and further to the cellular metabolite. The SimBiology files are available at this link:
Simulation Setup
There are four major processes that occur in our sensor setup as shown below:
which are the depolymerization of PET into TPA, the degradation of TPA to PCA, the transport of PCA and TPA, and the expression of GFP from the activation of transcription factors. The parameters of the TPA and PCA sensor systems are described below:
Using the SimBiology we created two sensor systems for our degradation analysis:
Through an extensive literature search, we found the enzyme kinetics parameters and protein concentrations as listed below. We converted all values of Vmax and kcat in units of M product / (s * M protein). We used non-reversible Michaelis-Menten as our enzyme kinetics parameters except the degradation of PETase, which we approximated using our zero order enzyme kinetics equation described previously and the transport of TPA and PCA by TpiAB and PcaK respectively, which followed a law of mass action kinetics.
We ran the simulation with the following parameters:
- “600 uM” of PET added in solution
- Constant enzyme concentrations
- Reversibility of reactions not considered
- Simulated rich oxygenated media with excess NADPH
- Fixed 50 uM of transcription factor concentrations (a gross exaggeration)
- 30 Day simulation
Simulation Results
In the PCA detection system, we predict that there are high levels of PCA in the media, but the rate limiting reaction in the degradation pathway is the conversion of DCD to PCA. Transport of PCA limits the activation of the PcaU. The model predicts that concentrations of PCA will exceed the solubility limit (120 uM in water), but we see that there is full activation of PcaU prior to the solubility limit being reached. There is rapid activation of PcaU at about 1.5 days.
In the TPA detection system we see an immediate increase in the concentration of TPA in the media, with a similar problem of the rate of transport limiting the activation of TphC. In this simulation, we see an increase in the time to full response to about 7 days, which is about 4x longer than the PCA based system. However, in this simulation we exceed the solubility limit of TPA (90 uM) rapidly, therefore we tweaked the model to a fixed concentration 90 uM concentration of TPA in the media.
1: Sabathé, F. & Soucaille, P. Characterization of the CipA scaffolding protein and in vivo production of a minicellulosome in Clostridium acetobutylicum. J. Bacteriol. 185, 1092–1096 (2003).
2: Yoshida, S. et al. A bacterium that degrades and assimilates poly(ethylene terephthalate). Science 351, 1196–1199 (2016).
Contact Us
umarylandigem@gmail.com
Biology - Psychology Building
4094 Campus Dr, College Park, MD 20742
umarylandigem@gmail.com
Biology - Psychology Building
4094 Campus Dr, College Park, MD 20742
© University of Maryland 2018
Follow Us
