PETase Control Circuit
Description: Wild type PETase was used as a control for our tests of the modified PETase enzyme. This circuit was put together with gibson assembly to add it to a J23110 constitutive promoter and the k1357009 amilCP reporter gene with a double terminator. The control PETase was used to compare the rates of the modified PETase to ensure that the modifications work correctly and faster than the wild type.A PELB tag was added to the PETase enzyme to excrete it from the cell so plastic degradation can occur properly.
Modified PETase Control Circuit
Description: This circuit is composed of a modified PETase (polyethylene terephthalate) enzyme that is mathematically modeled to work 20% faster than the original. The modifications made were to the active site to make it more like a cutinase active site which can work more efficiently because of two amino acid changes in the protein. The modifications involved S238F and W159H switches. These were brought up because the modifications made the active site similar to the Cutinase enzyme active site. The similarities in the structure of PET and Cutin show that the active sites of the enzymes can be very similar because of where the cuts are made, so trying to model the PETase to work more like Cutinase has strong support. With this part, we added a constitutive promoter and the k1357009 amilCP reporter gene with a double terminator. This circuit as compared to the wild type enzyme in the same circuit to compare the rates of reaction and prove that the modified version is actually faster.
MHETase Control Circuit
Description: The mHET circuit constructed contains an amilCP reporter gene with a double terminator, and a constitutive promoter so that the enzymes are constantly being made. The mutPETase enzyme will break down the PET polymer into its monomer mHET. The mHETase enzyme will then break down the mHET into its basic components of ethylene glycol and terephthalic acid, which can be broken down by normal metabolic pathways in the cell. With this circuit fully functional, the cells would be able to survive on the plastic as the main carbon source.