Team:Exeter/Design
Design
The Enzymes
PETase and MHETase are two naturally occurring enzymes that were discovered in 2016 by a team of Japanese researchers. They are produced by Ideonella sakaiensis and have the capacity to break down polyethylene glycol (PET) into its building blocks. This occurs as a two step process. First PET is broken down by PETase into mono-2-hydroxyethyl terephthalate (MHET), bis(2-hydroxyethyl) terephthalate (BHET) and terephthalic acid (TPA). Then MHET is further degraded by MHETase into TPA and ethylene glycol (EG), while BHET is degraded by BHETase into MHET. These reactions are represented in Figure 1. Since 2016 further research has been done into improving the activity of these enzymes, resulting in mutants with various higher rates of degradation. Our constructs are mainly based on these papers including both the wild type and the mutants. However, we have also produced novel constructs in two ways. Firstly we used the available literature to identify beneficial mutations and combined them in different ways to form novel constructs. Secondly, we made use of the method of ancestral reconstruction in order to engineer 4 novel constructs that we aimed to test and see if they showed any improved activity.
Plasmid Design
During the early stages of the project there were two ideas on how to introduce our enzymes into the filter system. We could either use pre-purified enzymes or engineer bacteria to produce the enzymes within the filter itself. To allow us to explore both possibilities we designed and built two sets of gene constructs for our project: one set of genes was to be expressed intracellularly and subsequently purified; the second set was intended for extracellular expression. The intracellular constructs were synthesised by TWIST as complete genes with the requirement that they did not contain the forbidden restriction sites listed by iGEM (EcoRI, XbaI, SpeI, PstI). These were cloned by TWIST into one of their stock plasmids pET28 (Figure 2a). Individual coding sequences, used to construct the genes for extracellular export were synthesised as linear fragments by IDT, and assembled, with promoter-RBS, terminator and a University of Exeter plasmid (pX1800) using the TypeIIS assembly method (Figure 2b).
All of the constructs were designed to be under the control of the inducible T7 promoter, which we have shown to be non-leaky. All of the coding sequences were designed to include a His-tag at the N-terminal of the resulting protein to allow for verification of protein expression by Western blot analysis and purification by Nickel Affinity chromatography when appropriate. The coding sequences we used were taken both from existing literature and from our own work in ancestral reconstruction, to produce a library of mutants of PETase, BHETase and MHETase. For extracellular expression two signal peptides were chosen, lamB and malE, based on previous literature work that demonstrated successful excretion of PETase from E. coli. Allowing for the potential uptake of MHET by E. coli, an ompA signal peptide was also added to the MHETase enzymes to target enzyme activity to the periplasm of E. coli.
As the project progressed, feedback from our Human Practices led us to refine our ideas. In particular, with his knowledge of environmental law, Dr David Santillo, Honorary Research Fellow and Greenpeace Scientist, advised us that it would be “far easier from the outset of creating a product just containing the enzymes - no bacteria.” Dr Santillo suggested that in a household washing machine bacteria such as E. coli. could not be present in the filter. Therefore after initial cloning of the constructs for extracellular expression no further experiments were performed with these constructs. Our work in the biology labs has since focussed on expression and purification of our intracellular enzymes.
Application
The aim of our project is to eventually integrate the enzymes into a real life application such as an enzyme based filter system that is capable of capturing microfibres coming off during washing cycles in washing machines and degrade them by secreting the set of enzymes we are using. As such, we designed different filter prototypes in order to test them and see which one is most successful in capturing the fibres and delivering the enzyme solution. The full list of designs we produced can be found on our Hardware page.
