Colorimetric detection of microplastics based on anchoring peptide-modified Au colloid

1. Expression and purification of anchoring peptides

In order to specially recognize microplastics, we utilized genetic engineering strategies to express anchoring peptides Cg-Def, TA2, and LCI. Many experiements have been done to optimize the design of recombinant plasmids, protein expression and purification of these three anchoring peptides. Fig.1A,1B and 1C gave the finally designed plasmid maps in which the target protein gene LCI, TA2 and Cg-Def was follwed by TEV site, His Tag and sfGFP (superfolder Green Fluorescent Protein), respectively. PCR results in Fig. 1D showed that both the target protein and sfGFP were successfully inserted into pET-30(a). SDS-PAGE results indicated that the fusion protein was successfully expressed and purified, as shown in Fig. 1E.

2. Affinity Assay of anchoring pepetide against plastics

To check the affinity capability of the expressed target peptide against various microplstics, we utilized fluorescence microscopy to measure the fluorescence emitted from sfGFP in the fusion protein. Meanwhile optical image was also done to show the plastics appearance. It was found from Fig.2A that when PS, PLA or PP was incubated with TA2 anchoring peptides, there occurred strong fluorescence emission on the plastic surface. Similar phenomenon was also observed when we used LCI and CG target peptides to mix various plastics (Fig.2B and 2C). In contrast, no fluorescence was detected in the case of PET, indicating that these anchoring peptides showed rather weak affinity to recognize PET. Here it should be noted that we also used sfGFP containing no anchor peptides as control, and no fluorescence emission was detected. These results suggest that we have successfully obtained anchoring peptides.

3. Detection of Microplastics based on anchoring peptide modified Au colloid

After we synthesized Au colloid by the reaction of HAuCl4 and sodium citrate, we added the anchoring peptide TA2 into Au colloid to make them to combine together. We optimized pH value and the ratio of Au colloid versus anchoring peptide to finally get the modified Au colloid for the next experiment.

3.1 RGB sensor-based detection results

Fig.3A gave the color changes induced by the addition of microplastics to the anchoring peptide modified Au nanoparticles solution. It was observed that when the concentration of microplastics was less than 2 ug/mL, Au nanoparticle remained red colour. As the concentration of microplastics increased, Au nanoparticle gradually aggregated in solution and displayed purple color. Here we used RGB sensors to measure the values of red light channel, green light channel and blue light channel. They are denoted as R-value, G-value and B-value, respectively. Next we used multiple linear regression model, and successfully find the linear relationship between the concentration of microplastics and RGB fitted data (Fitted_Data = -0.1172*R_values + 0.5765*G_values - 0.5871*B_values+29.0269). As seen from Fig.3B, the standard deviation is ~ 0.99, indicating that RGB method we designed here was accurate and feasible for the detection of microplastics.

3.2 UV-Vis Spectral measurement results

In addition to RGB sensor, we also used UV-Vis absorption spectrometer to detect the spectral changes. As seen in Fig.4A, the introduction of microplastics gave rise to the appearance of one new wide peak at ~ 700 nm. This phenomenon inferred that Au colloid aggregated to larger particle in the presence of microplastics. In order to avoid the spectral overlapping between 525 nm and ~ 700 nm, We selected the absorbance ratio of A750/A525 to describe the extent of gold nanoparticle aggregation. As shown in the Fig.4B, there was a linear relationship between A750/A525 value and PS concentration in the range of 0-10 μg/mL, with a linear regression correlation coefficient of 0.99. This result indicated that UV-Vis absorption method is promising for the quantification analysis of PS plastic concentration.

Conclusion and Outlook

In our project, we have successfully developed a simple, convenient and specific detection methods for microplastics. Instead of expensive Raman or FTIR instruments, we can use much cheaper UV-Vis absorption spectra or even RGB sensors to carry out quantitative analysis of microplastics. We firmly believe that in the near future, we can use our mobile phone to achieve the detection of microplastics in the enviroment. It will help the public to realize the occurance and damage of microplastics and then take actions to protect our earth.

PET Biodegradation

Taking into account that PET could hardly enter the E. coli cells, we hoped that PET-degrading enzymes could be displayed on the bacterial surface. To achieve this aim, here we used the membrane protein Lpp-OmpA. In order to ensure this protein could be expressed on the E. coli surface, we firstly constructed one plasmid pET-30(a)-Lpp-OmpA-sfGFP. Fig.5B and 5C showed the PCR results and fluorescence microscopic results. It was observed that sfGFP has been expressed on the surface of E. colicell. This result indicated that Lpp-OmpA could work in our system. Next, we constructed the degrading recombinant pET-30(a) Lpp-OmpA-Z1-PETase-MHETase plasmid. As demonstrated in Fig.6B, the PCR amplified bands appeared at the expected position, suggesting that this plasmid has been successfully constructed. Although we had tried to use 15% SDS-PAGE to detect the expression of both Z1-PETase and MHETase, there were no significant bands at the expected molecular weight. It might be because the targets protein was mainly expresed in the form of membrane protein. It is not easy to extract enough amount of membrane protein. Next we conducted HPLC detection to make sure if our engineered E. coli cells could degrade PET or not.

In our project, we used HPLC to detect the concentation of TPA product. Fig.7A showed the standard curve of TPA in PBS buffer (pH 7.45) . Next we measured if TPA was released following the 24h treatment of PET using the engineered whole-cell E. coli harbouring Z1-PETase and MHETase. As demonstrated in Fig.7C, TPA was successfully produced, indicating that Z1-PETase and MHETase had been successfully expressed and they could work together. We also carried out some optimization experiments (seen in Fig.7B), and finally found that the released TPA could reach 0.579 mM when pH and OD was adjusted to 9 and 20 respectively (seen in Fig.7C).

Conclusion and Outlook

In this work, we used one membrane protein Lpp-OmpA Tag to achieve the surface display of both Z1-PETase and MHETase fusion protein. Under this condition, these dual enzymes could effectively catalyze PET into TPA. The yield of TPA varied with pH and bacterial density. In the further work, we could optimize the ratio of these two enzymes or adjust the linker between them to achieve higher yield or higher stability.

Upcycling of TPA to vanillin

1. Plasmid Construction for TPA Upcycling Enzymes

In order to convert terephthalic acid (TPA) into the high-value compound vanillin, we first constructed one recombinant plasmid containing 4 genes, i.e. TphA1, TphA2, TphA3 and TphB. The former three genes encode the three subunits of terephthalate 1,2-dioxygenase (TPADO), and TphB encoded 1,4-dicarboxylic acid dehydrogenase (DCDDH). These two enzymes could catalyze TPA into protocatechuic acid (PCA). Next we constructed the second plasmid harbouring NiCAR gene and S-COMT gene, which encodes carboxylate acid reductase and catechol O-methyltranferase. The third plasmid was also successfully constructed, in which Sfp gene encoding phosphopantetheinyl transferase was incorporated into PACYDuet-1 vector. The enzymes are essential for the conversion of PCA to vanillin.

2.Protein expression

SDS-PAGE analysis revealed that several target proteins were successfully expressed in soluble form using E. coli BL21(DE3) as host cell. (Fig.9).

3.HPLC analysis results

Fig.10A showed the HPLC eluting profile of the supernatant collected following the addition of TPA to the E. coli intact cells co-expressing TPADO and DCDDH enzymes. HPLC comparison was also made using the standard sample of TPA and PCA under the same condition. It was found that PCA was produced successfully with the elution time of 7.068 min. The concentration of PCA was also calculated to be 0.078 mM.

Based on the standard curve we had done (data not shown). This result suggested that the first plasmid can work well. Next we detected if the second and the third plasmid could also work or not using HPLC method. As seen in Fig.10B, there appearred one small eluting peak at 14.279 min after the incubation of TPA with the intact E. coli celles which was co-transformed by plasmids piGEM24_06, piGEM24_07, and piGEM24_08. The injection of standard vanillin sample into HPLC gave rise to the appearance of the same eluting peak, indicating that we have successfully converted the reactant TPA to the product vanillin using the engineered whole cell. In this case, the concentration of vanillin was almost 0.031 mM by calculating using standard curves of vanillin.

Conclusion and Outlook

For the time being, we have successfully achieved the upcycling of TPA into vanillin using engineered E. coli coexpressing multiple enzymes. In further work, we need to continue optimizing various conditions, particularly focusing on enhancing the transmembrane transport of TPA, which aims to improve the yield of vanillin.

Gene editing to downregulate vanillin metabolism

In order to enhance the yield of vanillin, we carried out gene editing experiment to knock out one key enzyme YahK in the side pathway. Electrophoresis results showed that the target gene has been successfully knocked out, as seen from Fig.11C. More work needs to be done to check the effect of knoted-out gene on the yield of vanillin.

Conclusion and Outlook

In this part, we have successfully utilized a Cas9-homologous reorganization fusion system to achieve gene editing of E. coli BL21(DE3). In further work, more key genes relating to E. coli BL21(DE3) vanillin metabolism pathways need to be knocked out, downregulated or upregulated so as to significantly enhance the yield of vanillin.