Our Solution & Design
Welcome to iGEM 2021
Based on the ASTWS-China 2020 project, we hope to address the problems of incomplete degradation and insufficient efficiency of the original enhanced biofilm PETase plastic degradation system. We plan to adopt the enhanced biofilm composite dual-enzyme system (PETase and MHETase), and further optimize the enhanced biofilm expression of the membrane, PETase, and MHETase in order to realize the efficient and thorough decomposition of microplastics in water bodies.
1 Overview of our design
Last year, ASTWS-China 2020 had proved that the enhanced biofilm (by overexpressing the OmpR234 gene) could improve the degradation activity of PETase through the proximity effect between the substrate and the enzyme. However, just as shown in Figure 1, polyethylene terephthalate (PET) could be degraded to MHET and BHET by the individual enzyme PETase. To obtain a non-toxic and harmless final product, MHETase is introduced into our system. In brief, in our enhanced "plastic-eating" biofilm system, there involved two parts with enhanced biofilm system (OmpR234) and dual-enzyme degradation system (PETase and MHETase) (Figure 1). The synergy of the two systems can achieve the goal of adsorption of microplastics in water and the complete degradation of PET.
Figure 1 Overview of our project design.
MHET : mono-(2-hydroxyethyl) terephthalate
BHET : bis(2-hydroxyethyl) terephthalic acid
TPA : terephthalic acid
EG : ethylene glycol
2 Strengthen the biofilm system (BBa_K3576000)
In our project, we chose OmpR234 to regulate the production of biofilms, allowing the output of crimp monomers to form crimped fibers and biofilms to capture PET nano/microplastics which was proven to be effective by ASTWS-China 2020 (Figure 2). This part was constructed as part number BBa_K3576000. Click here [SS1] for more details.
Figure 2. Schematic diagram of the function of strengthening the biofilm system.
3 Dual enzyme degradation system
3.1 Strategy of the combination of PETase and MHETase
It is reported that the fusion of PETase and MHETase can improve the degradation activity of PET polymers. Based on this reference, we design two strategies for our dual enzyme degradation system, as shown in Figure 3.
Figure 3 Two strategies of dual enzyme degradation system.
Strategy 1 PETase-MHETase co-expression
At the very beginning of this iGEM season, our initial idea to combine PETase and MHETase is to co-express the two enzymes (Figure 4). However, after a background survey, we found that the fusion of PETase and MHETase was too unaffordable for the E. Coli expression system. Thus, after talking with our instructor, we temporarily abandoned this strategy.
Figure 4 Genetic circuit of co-expression of PETase-MHETase (BBa_K3906004).
Strategy 2 SPY (SpyCatcher-SpyTag) system
In order to make PETase and MHETase work together, the SPY system is introduced, including SpyTag and SpyCatcher. These two components are recombined with MHETase and PETase, respectively. Figure 5 indicates the genetic circuit design of the SpyTag-SpyCatcher system. PETase and MHETase complement each other through the non-covalent interaction of SpyCatcher and Spy-Tag, which is like a key to a lock to achieve specific binding so that the two enzymes merge together. In the following, we will use the abbreviation PETsae-SC and MHETase-ST for PETsae-SpyCatcher and MHETase-SpyTag, respectively.
Figure 5 (A) Schematic diagram of the combination of PETase and MHETase through the SPY system, and genetic circuit design of (B) PETsae-SpyCatcher and (C) MHETase-SpyTag.
3.2 PETase-SpyCatcher expression system (BBa_K3906000)
The detailed genetic circuit of the PETase expression system is shown in Figure 5-B. Here, we simply introduce PETase (BBa_K1582000) to give E. coli biodegradability of PET plastics. In addition, we connect SpyCatcher (BBa_K1159200) to it in order to work better with MHETase. The main function of this bio-brick is to overexpress PETase and degrade microplastics from the membrane.
3.3 MHETase-SpyTag expression system (BBa_K3906002)
The genetic circuit of the MHETase expression system is shown in Figure 5-C. We introduce MHETase ((BBa_K3039004), an enzyme that can decompose PETase's intermediate product of degradation (MHET) to TPA and EG, which ensure that the final product is non-toxic and harmless. Similarly, SpyTag (BBa_K1159201) part is also added to this expression circuit, to ensure combination with PETase-SC.
3.4 Double fluorescence reporting system
In order to visually observe whether the expression of PETsae-SC and MHETase-ST is successful, the eGFP and mCherry fluorescent reporter genes are introduced into the two plasmids (Figure 6). If the two combine as expected, we can see the change in fluorescent color. When separated, one is displayed in red and the other is displayed in green, and when combined, the two are displayed in yellow.
Figure 6 Genetic circuit design of double fluorescence reporting system, including two parts PETase-SpyCatcher-eGFP(BBa_K3906001) and MHETase-SpyTag-mCherry (BBa_K3906003).
1 Alexandre V. Pinto, et. al. Reaction Mechanism of MHETase, a PET Degrading Enzyme. ACS Catal. 2021, 11, 16, 10416–10428.
3 Brandon C K, Erika E, Mark D A, et al. Characterization and engineering of a two-enzyme system for plastics depolymerization. Proceedings of the National Academy of Sciences, 2020, 117 (41) 25476-25485.