Part:BBa_K3906002
MHETase-SpyTag
It is the key part that is responsible for expressing MHETase. The MHETase can hydrolyze MHET to TPA. In order to ensure that MHETase can be fully in contact with PETase, the sequence of coding SpyTag peptide was added after MHETase, which can be recognized by SpyCatcher peptide.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 332
Illegal PstI site found at 1093 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 332
Illegal PstI site found at 1093 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 332
Illegal PstI site found at 1093 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 332
Illegal PstI site found at 1093 - 1000COMPATIBLE WITH RFC[1000]
Usage and Results
1 Plasmid Construction
We designed our functional parts and cloning into pET-21a(+) backbone plasmid chemical synthesized by GenScript. As mentioned on our design page, strengthening the biofilm, PETase and MHETase expression is our main parts. To confirm the correctness of the plasmid, we used BamHI and EcoRI restriction enzymes to digest the plasmids. The gel electrophoresis results (Figure 1) showed that the OmpR234 (739 bp), PETase (1236bp), and MHETase (1821bp) genes were constructed as expected. In addition, we confirmed the results by sequencing the entire plasmid.
2 Protein expression test
SDS-PAGE electrophoresis was used to check the expression of OmpR234 protein, PETase proteins, and MHETase proteins. As shown in Figure 2, compared to the blank control, the lane contained MHETase(42 kDa) protein indicated that these three proteins have been successfully expressed.
3 Enzyme Activity Test of MHETase
We measured the concentration of TPA (MHET degradation product) by HPLC to analyze the degradation activity of MHETase. The result is shown in Figure 3, and the relationship between the concentration of product TPA and MHET is shown in Figure 4. By comparison, when the concentration of the substrate (MHET) is in the range of 0 - 2 mM, the results indicated that the co-expression of OmpR234 can improve the degradation activities of MHETase. However, when the concentration of the substrate (MHET) reached more than 2mM, the presence of OmpR weakened the MHETase degrading activity.
4 Real sample test
In order to verify the degradation effect of our system on the real PET plastic, we used HPLC to test the degradation effect of our system using PET power. The result is listed in Table 2 and figure 13. The results indicated that OmpR strengthen biofilm can significantly improve the degradation efficiency of the dual enzyme (PETase-MHETase). And compared to the calculated rate of reaction from model, although the calculated value is not exactly the same as the experimental value, but basically in line with the tested reaction rate, which further verify the MM equation predicts the measured data very well.
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