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Revision as of 01:18, 18 October 2016
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.
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.
