Part:BBa_K3906000
PETase-SpyCatcher
It is the key part that is responsible for expressing PETase. The PETase can hydrolyze PET to MHET. In order to ensure that PETase can be fully in contact with MHETase, the sequence of coding SpyCatcher peptide was added after PETase, which recognizes a cognate 13-amino-acid peptide (SpyTag). Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 195
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 PETase (30 kDa) protein indicated that PETase protein has been successfully expressed.
We evaluated the production of PETase using the constructed expression systems in the E. coli BL21 line. To compare the induction effects by IPTG addition, we also performed a time series analysis in our expression system. As shown in Figure 3, the expression of PETase in BL21 is time-dependent and increased with the IPTG addition. When we analyze the PETase by means of Western blotting using the concentrated culture media, we observed clear bands with a molecular weight of approximately 30 kDa from IPTG induced BL21 cells (Figure 3). As the calculated molecular weight of PETase is 30.2 kDa, we suspected that these bands were the expressed PETase.
3 Enzyme Activity Test of PETase
The enzyme activity of PETase was performed by p-NP assay which is a common way to quantify hydrolytic activity. We selected p-Nitrophenylbutyrate (p-NPB) as the substrate, which can be hydrolyzed by PETase to p-nitrophenol (p-NP), but cannot be hydrolyzed by MHETase (Figure 4). Thus, using p-NP assay, the enzyme activity of PETase and the impact of the addition of MHETase can be characterized by measuring the optimal optical density (OD) values at 405 nm. The plasmid used here is PETase-SpyCatcher (BBa_K3906000, Abbr. PETase-SC), OmpR234 (BBa_3576000, Abbr. OmpR), and MHETase-SpyTag (BBa_K3906002, Abbr. MHETase-ST).
As shown in Figure 5, after overexpressing by PETase-SC and OmpR, the OD405 value of p-NP gradually increased at different substrate concentration levels with the extension of reaction time, which indicates that the degradation activity of the PETase. While overexpressing the PETase-SC + OmpR protein and PETase-SC + OmpR + MHETase-ST, respectively, it can be seen from Figure 6 that there is no significant difference between the two groups. In other words, it demonstrated that the addition of MHETase-ST would not affect the enzyme activity of PETase.
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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