Team:Tianjin/Model
Heterogenous Degradation By PETase
Model Overview
In our experiment we use engineered bacteria as machines to secrete PETase to degrade PET.At first the bacteria secrete PETase ,and then enzymes diffuse into liquid phase body from the cell surface ,from liquid to the surface of PET successively.PETase adsorbs on PET during which process the substrate binding sites of PETase contact with the surface.Finally PETase finds catalytic sites on plastics and combine them with its active center.Ester bonds are broken and chains in PET are ruptured,resulting in the degradation of PET.
Assumptions
1. There exists feedback inhibition regulation in the growing process of a single bacteria. Unlimited growth of population growth can’t be supported due to the limitation of space and resources in a certain environment. When the population of individual bacterium has too much increased, the environment degrades and the average resource share declines, resulting in reduction in birth rate while mortality rate is increasing. Consequently it is reasonable to assume that there exists feedback inhibition regulation due to the influence of environmental factors.
2. A cell can be roughly considered as a sphere.
3. Enzymes are in the dynamic equilibrium when transferring in the liquid phase.
4. The resistance of enzymes’ spread from liquid body to the surface of PET is much larger than that of from the cells’ surroundings to liquid body.
5. It takes several steps for the enzymes to complete the degradation of PET. Enzymes have the substrate binding sites and active centers. We assume that the enzymes are firstly combined with the polymer substrate through their own substrate binding sites, and then the active centers catalyzed the degradation of the polymers.
6. The degradation of PET takes place on the surface of PET.
7. The mass transfer of enzymes in the liquid phase will cause some of them to stay in the liquid body and the delay of enzyme concentration changes on the PET surface.
1. Reporting System
The basis of our reporting system is the part BBa_K339007, Designed by Emily Hicks from Group iGEM10_Calgary. This part can sense the CpxR protein, which will form spontaneously in E.coli when inclusion body and misfolding protein present in the periplasm of E.coli, and then start expressing RFP so that we can detect red fluorescence. As we all know, the inclusion body will inevitably form when we overexpress heterologous protein like PETase in E.coli. Therefore, the emission of red fluorescence can report the overexpression of PETase. What is more, this device can be modified to report overexpression of any heterologous protein only if the PETase gene is replaced by another heterologous gene. After the red fluorescence is detected, we could start the purification of protein.
2. Cell Lysis Based Regulation System
The regulation system consists of two section. The first section is based on the already mentioned reporting system. We change the RFP gene to the novel ddpX (D-alanyl-D-alanine dipeptidase) gene from E.coli genome. The ddpX gene can hydrolyze the D-Ala-D-Ala structure in peptidoglycan molecule and cause damage to the cell wall of E.coli. Under normal condition, this gene only express when the cell is in starvation mode in order to use hydrolysate alanine as carbon source. However, if we overexpress this gene, the cell wall will be dissolved and finally cell lysis will happen. Therefore, in this system, when the PETase is overexpressed, the spontaneously forming inclusion body will induce the expression of ddpX and cause cell lysis. It will provide us with a novel and convenient and way of protein purification when you use E.coli as chassis.
