Part:BBa_K3576003
eGFP-PETase
1
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 97
Illegal NgoMIV site found at 123 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 986
Illegal BsaI.rc site found at 787
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 biofilm part and PETase expression part are our two main parts. To confirm the correctness of the plasmid, BamHI and EcoRI restriction enzymes were used to digest the plasmids. The results of gel electrophoresis (Figure 1) shown that the lengths of OmpR and PETase genes are around 1500-2000 bp, respectively, which meets the expectation. Besides, we then confirmed the results by sequencing the whole plasmids.
To shorten the distance between PETase and the substrates, we introduced the Spy system, including SpyTag and SpyCatcher. These two components are recombined with PETase and OmpR parts, respectively. Figure 2 shows the genetic circuit design of the SpyTag-SpyCatcher system. To visually observe whether the expression is successful, GFP and mCherry fluorescent reporters were introduced into the two plasmids (Figure 2). In the following, we will use the abbreviation OmpR-SpyTag and PETase-SpyCatcher. If the two were bound as expected, we could see the change of fluorescence color. When separated, one appears red and the other appears green, and when combined, the two appear yellow. Therefore, we further constructed the corresponding plasmid and carried out electrophoresis verification.
2 Recognition Verification of Spy System
To verify the recognition of OmpR-SpyTag and PETase-SpyCatcher, we transformed three sets of plasmids (OmpR-SpyTag, PETase-SpyCatcher, OmpR-SpyTag + PETase-SpyCatcher) into E. Coli and observed them with a fluorescence microscope. The results are shown in Figure 3. As mentioned above, E. Coli transformed with OmpR-SpyTag was designed with mCherry reporter which display red (Figure 3-B) in micrograph and PETase-SpyCatcher designed with GFP reporter display green (Figure 3-A). It is worth mentioning that when the two expressed together, the microscopic image shows yellow, which also qualitatively proves the effective combination of OmpR-SpyTag and PETase-SpyCatcher.
3 Degradation Activity Test of Co-expressed System
In addition, the p-NP assay was used to further test the effects of the strengthened biofilm and Spy system on the degradation activity of PETase. PETase-SpyCatcher and PETase-SpyCatcher+OmpR-SpyTag plasmids are transformed into E. Coli BL21 and overexpressed respectively. Then these bacteria solutions are mixed with p-NPB substrates and measure the absorbance at the wavelength of 405 nm. Figure 4 demonstrated that, with the extension of time, the OD405 value increases. At the same time, it is significant that the OD405 value of the co-expression system is higher than that of PETase alone. In other words, with the help of Biofilm and Spy system, the degradation activity of PETase could be improved. After discussing with our instructors, the proximity effect between the substrate and the enzyme may be one of the reasons for the increased degradation efficiency.
4. Real Sample Degradation Test
In order to verify the degradation effect of our system on the real PET plastic, we cut the PET plastic bottle and grind it to the microplastic. After expressing our ultimate plasmid and culturing it on plastic fragments, we can see the adhesion of biofilm (Congo red staining test) on the large plastic fragment (Figure 5-B inserted). On the other hand, our simulated microplastics were mixed and cultured with our engineered bacteria, and the concentration of MHET (Mono-(2-hydroxyethyl) terephthalic acid), the PET degradation product, in the solution was measured by HPLC. The results are shown in Figure 5-C. The relationship between peak area from HPLC results and MHET concentration was plotted to further analyze the degradation efficiency, shown in Figure 5-(A-B). Figure 5-A showed the standard curve of the MHET standard. The results show that there is good linearity between MHET concentration and peak area with R¬2 = 0.999. Under the same experimental conditions, the engineered E. Coli overexpressed with PETase-SpyCatcher and co-expressed with PETase-SpyCatcher and OmpR-SpyTag was also performed to degrade the real PET microplastics. LB medium and unmodified E. Coli solution are set as the control groups. The results shown in Figure 5-B are revealed that the peak area obtained by the co-expressed group (PETase + OmpR) is significantly (approximately 1.66 times) higher than that of the PETase alone group.
In summary, these results further illustrate that shortening the distance between the enzyme and the substrate is a reliable solution to improve the degradation efficiency of PETase.
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