The DNA coding for the 6 enzymes required for breakdown and assimilation of PET was too long to fit on one plasmid. To rectify this and to be able to test smaller subsystems, the PETase and MHETase genes were placed on Backbone 1, Plasmid 1. The Glycolaldehyde Reductase, Glycolaldehyde Dehydrogenase, Glycolate Oxidase, and Malate Synthase were placed in sequence on Backbone 2, Plasmid 2.

Plasmid 1

Plasmid 1 uses an AraC and pBAD promoter to regulate expression of PETase and MHETase. The transcription factor made from AraC usually binds and represses the pBAD promoter, halting transcription of the plasmid. The inducer, Arabinose, can be added to the media, and this molecule binds to the AraC transcription factor on the DNA strand and changes its conformation so that transcription can occur [1]. The reaction scheme on the left explains a more complete mechanism of the transcription/translation of these proteins. The creation of AraC protein is related to a constitutive promoter which we assume enters the system as a constant rate, K. This method was also used by the UC Davis iGEM team in 2012. We assumed a fast-equilibrium hypothesis on the formation of the dimer and an essentially constant pool of arabinose in the environment. We can also streamline the binding of the two arabinose inducers to the AraC dimer into one reaction determined by the rate parameters k3+ and k3-. Since the amount of RNA polymerase does not change relative to these molecules, and since the frequent assumption used in literature is to group the transcription and translation rate into one overall rate of protein production, we simplify the system further. From these assumptions, we can simplify the system down into the system shown on the right.

Model Equations:

https://bmcsystbiol.biomedcentral.com/articles/10.1186/1752-0509-5-111 useful description and numbers for AraC promoter

Simulations:

Table 1: List of values used in simulation parameters

We simulated 12 hours of time with the genetic system, and this figure maps out the expression levels of the free repressor protein AraC and Plasmid 1 genes, PETase and MHETase. The initial level of AraC was set to 40 monomer units per cell, known from [source]. The population of cells was 1e+08. The expression of this genetic system under these conditions shows the objective proteins are high while free AraC protein, which would normally suppress expression, is relatively low. If MHETase and PETase levels were overlaid, they would be nearly identical. The only difference is that MHETase decays slightly slower than PETase and reaches a higher maximum concentration.