Results

Content

Enzyme production Plastic depolymerization Protein production Future perspective

Enzyme production

We successfully cloned the gene encoding for FAST-PETase into the pRSFDuet-1 vector, using restriction digestion and ligation methods. We verified the correct insertion of the gene by restriction analysis and Sanger sequencing. We then transformed the recombinant plasmids into an expression strain. The expression was induced via IPTG and the pellet and supernatant of the expression were analyzed using SDS-PAGE.

PCR

After multiple failed attempts at cloning, we had to perform PCR amplification for the MHETase insert. It was first amplified via PCR using DreamTag polymerase (Thermo Fisher, #EP0703) and purified using GeneJet Gel Extraction Kit by Thermo Scientific. The purified PCR product was then analyzed by running electrophoresis on a small amount with 1% agarose gel as shown in Figure 1.

Figure 1. MHETase construct successfully amplified. The desirable size of the construct is expected to be of 1930 bp.

PETase and MHETase transformation

The constructs for FAST-PETase and MHETase were introduced into competent TOP10 E. coli via electroporation. The transformed colonies for both enzymes showed growth in LB plates supplemented with kanamycin as shown in Figure 2.

Figure 2. Growth of the E. coli cells transformed with the constructs on the kanamycin-supplemented plate. Left: MHETase constructs in TOP10, Right: FAST-PETase constructs in T7.

Restriction analysis

To confirm that the colonies were bearers of the correct construct, a restriction analysis was carried out. The plasmid was first purified with a GeneJET Plasmid Miniprep Kit (Thermo Fisher, #K0503). Next, the plasmid was digested using the restriction enzymes NdeI and XhoI (both Thermo Fisher, #ER0581 and #ER0692, respectively) (35 minutes 37ºC for the reaction and 5 minutes 80ºC to stop the reaction). The products of the restriction were analyzed by running electrophoresis in a 1% agarose gel as shown in Figure 3.

Figure 3. Plasmids purified from the colonies restricted with the NdeI and XhoI enzymes. Left: FAST-PETase-pRSFDuet1 restriction. Right: MHETase-pRSFDuet1 restriction. The size of the pRSFDuet-1 backbone is expected to be 3775 bp and the size of the FAST-PETase insert should be 888 bp, while MHETase should be 1872 bp. The results of the left gel picture correspond to these sizes, but for MHETase the insert size is too small.

Sequencing

After restriction analysis suggested that our FAST-PETase insert had been successfully cloned into pRSFDuet-1, we purified products from some of our colonies and sent them out to Eurofins Genomics for sequencing. Using the Sanger sequencing results obtained by sequencing with the DuetUP2, and T7term primers, we found that the sequence within our multiple cloning site perfectly matched our expectations as shown in Figure 4.

Figure 4. MCS-2 of pRSFDuet-1 was sequenced once in both directions, with both reads perfectly matching the sequence of BBa_K4701202 as denoted by the vertical red lines, and the surrounding sequence from the backbone.

Enzyme expression and secretion

When we had the confirmation that the expression vectors were good, we initiated protein expression by adding IPTG into the T7 liquid culture on LB-broth with OD600 of 1. The expression lasted 24 hours until the broth supernatant and pellet were collected. These were used to run an SDS-PAGE shown in Figure 5.

Figure 5. SDS-PAGE gel with pellet and supernatant samples after 24h protein expression. FAST-PETase should be 30.731 kDA. There is a band around that size in each sample, including the supernatant, which could indicate that the enzyme is being secreted into the growth media. Though without a proper control, the result is inconclusive.

Plastic depolymerization

We performed an experiment of plastic depolymerization by adding chemically and thermally pre-treated commercial plastic and gf-PET film (GoodFellow, ES30-FM-000250) into the growth media of the expression host. They were left at 37ºC for 5 days. The plastic samples were weighted before and after incubation. Unfortunately, no loss of mass was observed, most likely due to the issues we had with enzyme secretion.

Protein production

Growth experiments on TPA and EG

We successfully grew some common soil microbes on the M9 minimal medium supplemented by 30 mM of TPA and on the M9 minimal medium supplemented by 10 g/L of EG, as shown in Figure 6. P. putida and R. opacus were growing on both mediums. Meanwhile, C. testosteroni was only able to grow on minimal media containing 30 mM of TPA. This result indicated that TPA and EG obtained from enzymatic depolymerization could be utilized as carbon sources for microbial growth.

Figure 6. Growth of P. putida, R. opacus, and C. testosteroni in the presence of PET monomers, respectively, from left to right. a) PET + 30 mM of TPA, b) PET + 10 g/L of EG.

To produce the protein-rich biomass, R. opacus, and C. testosteroni were cultivated in the M9 broth medium containing TPA and EG as seen in Figure 7. Due to the time constraints, we were not able to execute similar experiments with P. putida

Figure 7. The biomass obtained from the microbes grown on the M9 broth medium supplemented by TPA and EG.

Metabolism and Bioconversion of PET monomers

Data obtained by HPLC analysis shows that PET monomers can be utilized as a carbon source to produce biomass. This can be seen as the decrease in TPA concentration over time in the biomass of R. opacus as shown in Figure 8. On the other hand, TPA was not metabolized by C. testosteroni. Due to the time constraints, the metabolism of EG by the strains was not studied with HPLC.

Figure 8. The metabolization of TPA by R. opacus and C. testosteroni was analyzed by HPLC.

Protein production

Lastly, we found that protein-rich biomass can be produced by growing microbes with PET monomers as the primary carbon source. The protein content of the resulting biomass was analyzed with a Bradford protein assay. We found that R. opacus successfully produced protein as shown in Figure 9. Even though we did not manage to analyze the protein content of biomass produced by C. testosteroni, the result from R. opacus indicates that producing single-cell proteins from PET monomers is a viable strategy.

Figure 9. Protein produced by R. opacus cultivated on M9 minimal medium containing 30 mM of TPA was assessed using Bradford analysis.

Future perspective

Embarking on our iGEM journey, we knew that we had a very ambitious project, with multiple stages which were not really the core knowledge of any of our team members. However, as we were all interested and motivated to prove that our concept could be viable, we put our best effort towards it. Due to time constraints, our team was unable to complete many of the experiments and protocols planned, so here is a list of experiments our team would potentially work on in the future.

Firstly, we should work on MHETase cloning and expression, to improve the degradation as PETase and MHETase work in synergy to completely depolymerize PET into TPA and EG. After that, many different analyses could be done to measure the enzyme activity and degradation rate.

In terms of protein production, we did not have time to engineer P. putida or E. coli with our constructs. A far future prospect would be to produce specific amino acids and nutritional substances in the cells. In addition, a comprehensive analysis of the biomass obtained needs to be carried out to ensure that the protein produced is safe.

We see great prospects in the project if there was more time and knowledge to execute all of our experiments. But hopefully, future iGEM teams or even other research groups will be inspired to work on similar projects.