Team:UMaryland/TPAvPCA
TPA vs PCA Detection
a consideration of response time and sensitivity
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 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.
In the fixed TPA concentration model, we see an even longer increase in the time to full response to almost 30 days, which is now 20x slower compared to the PCA based system. Therefore we concluded that for fastest response time to degradation of PET, a PCA based sensor system would be much better than a TPA based sensor system.
With regards to sensitivity, we were unable to get a clear picture from our literature search. We searched for lacZ activity which is downstream of the PCA or TPA activated promoter system. There are conflicting units of specific activity (U) and Miller Units, which cannot be converted between each other. However, we found that the detection limit of PCA is close to 1 uM while the detection of TPA is close to 10 uM [8,10]. Therefore we concluded that the sensitivity of the PCA sensor is also better than the TPA based sensor.
Simulation Conclusions
- The biochemistry literature is of limited help when trying to acquire values
- PCA Detection System provides better response time and sensitivity vs.the TPA Detection System
- Transport across the membrane the limiting factor in both scenarios
- Increasing the expression of transport proteins the biggest challenge
- PcaK transport system is much faster and smaller than the TpiBA system
- Metabolism of PCA negligible in limiting response of PcaU system
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umarylandigem@gmail.com
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