Team:ASTWS-China/Engineering

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Summary

We have successfully constructed and tested four main composite parts that are important to our project. The strengthening biofilm shortens the distance between PETase and nano/microplastics, and the fluorescent protein reporting system. The co-localization of PETase and enhanced biofilm is also successfully proved by the results of the fluorescent photo. Besides, p-NP assay results revealed that the proximity effect between enzyme and substance could improve the degradation efficiency of PETase.

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, which meets the expectation. Besides, we then confirmed the results by sequencing the whole plasmids.

Figure 1 Nucleic acid gel electrophoresis results of OmpR234 and PETase.roduction, use, and fate of polymer resins, synthetic fibers, and additives (1950 to 2015; in million metric tons)[2].

2. Protein expression test

SDS-PAGE electrophoresis was used to check the expression of OmpR234 and PETase proteins. As shown in Figure 2, compared to the blank control, the lane contained OmpR234 protein (27 KDa) and PETase (40 KDa) protein indicated that these two proteins have been successfully expressed.

Figure 2 Protein SDS-PAGE electrophoresis results of OmpR234 and PETase.

In our strengthening biofilms system, as shown in Figure 4, a strong T7 promoter (BBa_I712074) and strong RBS combination (BBa_B0034) to maximize protein production, and a double terminator (BBa_B0015) to end transcription. Therefore, by overexpressing OmpR234 (BBa_K342003)​, biofilms could become much more sticky and easy for capturing nano-/micro-plastics.

3.1 Expression of OmpR234 Increases Biofilm Formation

To verify whether the insertion of the OmpR234 gene strengthens the production of biofilm, we mixed a commonly used dye, Congo Red (CR), with samples in a 2 mL-system, transferring them to 24-well microtiter plates and incubating with the glass cover for 3 days. If biofilms were present, they would absorb the dye and appear as red, so the strengthened biofilm production would show a higher concentration of dye.
As shown in Figure 3, compared with the control groups, LB medium and E. Coli (BL21), the third group which overexpressed OmpR234 demonstrates a stronger red color, which indicated higher production of biofilm.

Figure 3 Congo red assay stains biofilm after overexpressing of OmpR234 (BBa_K3576000).

3.2 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 (pNPB) as the substrate, which can be hydrolyzed to p-nitrophenol (pNP) (Figure 4-A). The concentration of pNP can be measured by the characteristic absorption at 405 nm.
As shown in Figure 4-B, with the extension of reaction time, the OD405 value of p-NP gradually increased, which indicates that the degradation activity of the PETase.

Figure 4 (A)The mechanism of pNPB degradation; (B) OD405 of pNPB hydrolysis by overexpressed PETase.

4.1 Plasmid construction

To co-localized the PETase and OmpR234 enhanced biofilm, we introduced the double fluorescence reporting system, including GFP and mCherry. These two components are recombined with PETase and OmpR parts, respectively. Figure 5 shows the genetic circuit design of the GFP-mCherry reporting system. To visually observe whether the expression is successful, GFP and mCherry fluorescent reporters were introduced into the two plasmids (Figure 5). In the following, we will use the abbreviation OmpR-mCherry and eGFP-PETase. If the PETase and enhanced biofilm were co-expressed 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.

Figure 5 Genetic circuit design of double fluorescence reporting system

4.2 Co-expression Verification of Double Reporting System

To verify the recognition of OmpR-mCherry and eGFP-PETase, we transformed three sets of plasmids (OmpR-mCherry, eGFP-PETase, OmpR-mCherry + eGFP-PETase) into E. Coli and observed them with a fluorescence microscope. The results are shown in Figure 6. As mentioned above, E. Coli transformed with OmpR-mCherry was designed with mCherry reporter which display red (Figure 6-B) in micrograph and eGFP-PETase designed with GFP reporter display green (Figure 6-A). It is worth mentioning that when the two expressed together, the microscopic image shows yellow, which also qualitatively proves the effective co-localization of PETase and enhanced biofilm.

Figure 6 Fluorescence micrograph of engineered E. Coli transformated with (A) eGFP-PETase, (B) OmpR-mCherry, (C) and OmpR-mCherry + eGFP-PETase.

4.3 Degradation Activity Test of Co-expressed System

In addition, the p-NP assay was used to further test the effects of the strengthened biofilm on the degradation activity of PETase. eGFP-PETase and OmpR-mCherry + eGFP-PETase 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 7 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 enhanced biofilm, 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.

Figure 7 OD405 of pNPB hydrolysis by overexpressed PETase and PETase+OmpR

5. 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 9-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 8. The relationship between peak area from HPLC results and MHET concentration was plotted to further analyze the degradation efficiency, shown in Figure 9.
Figure 9-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 eGFP-PETase and co-expressed with eGFP-PETase and OmpR-mCherry 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 9-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.

Figure 8 HPLC results of MHET standard and real PET fragment degradation.

Figure 9 (A) Standard curve of MHET standard; (B) HPLC results of MHET concentration in real sample test (inserted is photo of biofilm (Congo red staining test) on the large plastic fragment).

In summary, these results further illustrates that shortening the distance between the enzyme and the substrate is a reliable solution to improve the degradation efficiency of PETase.

Future Work

Due to the COVID-19 epidemic, our experimental time is relatively limited this summer. However, according to the research, the thermal stability is another concern we need to consider. Normally, enzymes like PETase cannot survive in high-temperature surrounding and therefore not being able to degrade PET efficiently. Besides, to ensure the biosafety and convenience to use in real world, we also plan to purify the protein to better demonstrate its degradability (See more details in our Implementation page).
By collaborating with another team (TJUSLS_China 2020), we know that the thermal stability improvement of PETase is on the way, sometimes, the stability can be improved to be stable even above 75 ˚C. We have sorted out the references for improving the degradation efficiency and thermal stability of PETase. Our next goal is to build a PET degradation system with higher degradation efficiency and higher thermal stability. Hoping our world is better and safe. Our mission is still on the way.