Initial design

How can we solve the problem of plastic pollution impacting the environment and humans?

Our team was inspired to tackle plastic pollution after we learnt that plastics could contribute to endocrine disruption. After some research, we decided to focus on PET. We found that a popular solution was recycling because it prevents waste from entering the environment. However most recycling results in pollution of its own, and has other problems like scalability. We decided to focus on biorecycling, as the use of enzymes has fewer environmental impacts, and can produce the monomers of PET. These monomers can then be extracted and used for future PET production instead of raw materials, resulting in a circular economy for PET.

However, the PET biorecycling industry is mostly done on a small scale, and our interview with Georg Snyman found a large problem with public buy-in to recycling programs. We decided to focus on investigating ways to increase the enzymes efficiency and production, and therefore, the efficiency of the industry so that it could become more economically viable and contribute better to the downstream problem of plastic pollution prevention.

What genes could we use to achieve PET degradation?

In order to achieve this aim we researched different PET enzymes. We initially focused on waxworms due to an article we had found on them producing an enzyme to degrade PET, but soon found that it was infeasible due to the withdrawal of the gene sequence from publication. Consequently, the team searched for PETase enzymes in various microorganisms, including Ideonella sakaiensis bacteria, which had the entire PETase gene sequence published.

PET plastic degradation pathway is well-studied, and according to several studies by Japanese research groups, Ideonella sakaiensis bacteria contain two enzymes that relate to our project: PETase and MHETase. PETase breaks down the PET polymer to form MHET, TPA, and BHET, which MHETase further degrades into TPA and ethylene glycol.

A study conducted by Japanese researchers in 2016 demonstrated that IsPETase has a higher tolerance to environmental changes, such as variations in pH and temperature while maintaining its catalytic activity. The researchers compared IsPETase with different types of enzymes also degrading PET plastic from other microorganisms, such as fungal and bacterial cutinases at a reaction temperature of around 40 °C. Among these, IsPETase exhibited the highest PET plastic degradation efficiency (Yoshida et al, 2021)

Our team also checked previous iGEM teams' projects related to PET plastic degradation, and the 2018 Yale University iGEM team. “Making bacteria eat plastic" got the team’s attention (Yale iGEM). The Yale iGEM team also used the PETase gene for their project.

After many studies and experiments, several characteristics make IsPETase a promising candidate for the degradation and recycling of PET at higher tolerance to environmental changes. IsPETase has been extensively studied, and its entire gene sequence has been published. Our team can use this information to identify the IsPETase gene and request its synthesis from Mission Biotech company.

How can we increase the efficiency of biorecycling to make it a more viable solution to plastic pollution?

In order to enhance the PETase activity we did further research to see if we could focus on specific mutations.

Japanese scientists did investigations and showed that by altering 3 amino acids, serine 121 switched to glutamic acid, aspartic acid 186 switched to histidine, and arginine 280 switched to alanine, at the catalytic site on the PETase enzyme, (IsPETase^{S121E/D186H/R280A}), enhances the PETase activity compared to the wild-type PETase. Several papers demonstrated that different amino acid mutations on the IsPETase enzyme enhanced catalytic activity(Sevilla et al, 2023).

This mutated enzyme not only improves PETase activity but also retains catalytic efficiency at higher temperatures, accelerating PET plastic degradation (Brett et al, 2021; Maity et al, 2021; Son et al, 2019)

Our team wanted to upgrade the PET plastic degradation project to make an enhanced PETase enzyme with 3 amino acid mutations that we found to be promising while doing research on PET degradation utilizing a strain of Ideonella sakaiensis 201-F6, IsPETase^{S121E/D186H/R280A}. Our team also upgraded the original Yale project using bacteria for induction only.

How could we increase the amount of enzyme being produced?

We not only researched potentially more efficient enzymes but also ways to increase enzyme production. We found that scientists engineered yeast Gal1, and Gal10 promoter into a single Gal1,10 promoter on a plasmid for synthetic cloning that switches on in the presence of galactose and switches off in the presence of glucose. Research showed that the pGal1,10 promoter for the yeast strain, Saccharomyces cerevisiae, can induce 20-30-fold induction in the presence of galactose (Rajeshkannan et al, 2022). Therefore, our cloned IsPETase (BBa_K5094002) along with several mutations, expressed downstream of the pGal1,10 promoter (BBa_K5094001), could result in a 20-30 fold induction in the presence of galactose.

For galactose-inducing pGal1,10 promoter, this system is sufficient/efficient to induce 20-30 fold of the gene expression, which is one of the major benefits of using yeast our team designed. Our team did yeast transformation plate selection, and only yeast containing our pGal1, 10 promoter plasmid with G418 resistant gene can survive (more detail is on the cloning design cycle). However, if the team integrates the IsPETase gene in the yeast genome, respectively, G418 will not be needed for the selection anymore.

Can we isolate the enzyme to create a product?

We researched the idea of being able to extract the enzyme to be able to sell it to industry. The plasmid our team used for making bio-bricks was the pRSET A, a type of common prokaryotic expression plasmid, designed for high-level prokaryotic expression due to the T7 promoter. It encoded an N-terminal 34aa precursor peptide with a 6-histidine tag sequence to bind to the nickel-resin column for the in vitro protein purification. The bio-bricks our team designed were T7-IsPETase, T7-IsPETase^Thr116A, T7-IsPETase^{Thr116A/k259Glu}, T7-IsPETase^{Thr116A/M154Thr}, which T7 RNA polymerase specifically recognizes this promoter in bacterial competent cells of BL21(DE3) and our team expressed those variable IsPETase enzymes downstream of the T7 promoter in the presence of the inducer isopropyl β-D-1-thiogalactopyranoside (IPTG), respectively. This would allow the PETase to be extracted.

How can we create the desired mutations on the PETase

To verify our approach of generating pGal1,10-IsPETase (BBa_K5094010) mutations, the team cloned the wild-type IsPETase gene downstream of the pGal1,10 promoter plasmid (the same as the T7 promoter). Our team used this pGal1,10 promoter driving IsPETase gene in vivo to leverage that yeast-expressed under pGal1,10 inducible promoter can generate 20-30 fold induction in the presence of galactose(Rajeshkannan et al, 2022).

Here's the procedure our team did to generate several mutations in the IsPETase gene:

1. Cloning: The wild-type IsPETase gene was cloned downstream of the pGal1,10 promoter plasmid.
2. Site-Directed Mutagenesis: our team performed site-directed mutagenesis to introduce the desired mutations (S121E, D186H, R280A). Primers containing the mutant nucleotides were designed for this purpose, however, our team designed 18-20bp of complementary sequence on either side of the desired mutation (usually 1-3 mismatched bases), which should be sufficient for the primers to anneal to the pGal1,10-IsPETase plasmid during the PCR reaction. PCR: PCR was performed using the pGal1,10-IsPETase plasmid as the DNA template. DpnI Digestion: The PCR product using primers containing S121E mutation was treated with DpnI enzyme to digest the parental DNA template that contains methylation markers. The new mutant plasmid, pGal 1,10-IsPETase^S121E, remained undigested due to the lack of methylation. Bacterial Transformation: The mutated plasmid was transformed into bacteria to generate more copies of the pGal 1,10-IsPETase^S121E plasmid.
3. Repeating Mutagenesis: The team repeated the site-directed mutagenesis twice to introduce the additional mutations, with the aim of creating the enhanced IsPETase^{S121E/D186H/R280A} enzyme downstream of the pGal1,10 promoter.

Our team also did the same procedure to create the T7- IsPETase^{S121E/D186H/R280A} enzyme. After site-directed mutagenesis, our team sent out those mutant plasmids to sequence.

What problems did our team struggle with after doing site-directed mutagenesis to generate several mutant IsPETase enzymes downstream of either the T7 promoter or the pGal1,10 promoter?

When we received our sequencing, it showed that our desired mutations had not been successful. With Dr. HsiaoFen Han’s help, we noticed the IsPETase gene sequence contained multiple DNA fragments with repeat regions that our team didn’t expect while designing those primers, leading to challenges for the site-directed mutagenesis technique. The primers containing mutated nucleotides that our team desired to switch specific amino acids targeted incorrect amino acids due to these repeats so our team created T7-IsPETase (BBa_K5094006), T7-IsPETase^Thr116A (BBa_K5094007), T7-IsPETase^{Thr116A/k259Glu} (BBa_K5094008), T7-IsPETase^{Thr116A/M154Th}r (BBa_K5094009) instead. For the pGal1,10 promoter, our team didn’t make any mutations on the IsPETase gene, so the team has pGal1,10-IsPETase (BBa_K5094010), and pGal1,10-eGFP used as the control (BBa_K418008).

Our team still decided to use the IsPETase mutations downstream of the T7 promoter to do a functional assay because the mutations are also involved in the PETase catalytic activity. Other publications showed altered several amino acids in the IsPETase gene, to enhance the enzyme activity, our team predicted the mutations created by accident might also affect the catalytic activity (Sevilla et al, 2023).

We also decided to continue with tests for the pGal1,10-IsPETase, which the sequencing showed was successfully cloned, with pGal1,10-eGFP used as the control(BBa_K418008) to determine if the promoter was, in fact, able to increase expression of the enzyme.

What could our team do to fix the random mutations created on the IsPETase gene via the site-directed mutagenesis technique?

The team used pGal1,10-IsPETase plasmid as a DNA template to make mutations but failed due to the large size of the pGal1,10-IsPETase plasmid (~7kb). Several DNA fragments being repeated in the IsPETase gene sequence hindered the site-directed mutagenesis technique to alter the specific amino acids our team desired. Dr. HsiaoFen Han suggested that one way our team could do this is to directly add the mutations to the IsPETase gene sequence and order the synthesized mutant gene from the Mission Biotech company. After that, the team could do the traditional cloning as the team did with the wild-type IsPETase gene.

The team could also optimize several primer sets of annealing conditions further for site-directed mutagenesis.

What functional assays did the team perform to determine functionality for the yeast containing cloned genes downstream of the pGal1,10 promoter and DE3BL21 bacteria containing cloned genes downstream of the T7 promoter?

In order to determine if the plasmids were functioning within the E. coli and yeast we designed various functional assays, our team transformed the T7-IsPETase plasmid into DE3BL21 bacteria along with several respective mutations of the IsPETase plasmids, and induced the expression in the presence of IPTG. The pGal1,10-IsPETase plasmid was transformed into Saccharomyces cerevisiae yeast strain, as was the pGal1,10-eGFP as a control, and expression was induced in the presence of galactose to generate more IsPETase catalytic activity.

mRNA Production

For the functional assays, in order to determine if the mRNA is being produced, the team did RT-qPCR to detect IsPETase mRNA level after being induced by galactose for the pGal1,10 promoter in yeast.

To manipulate our team’s wild-type of the IsPETase enzyme with the control eGFP cloned downstream of the pGal1, 10 promoter, respectively, yeast containing pGal1,10-eGFP was the control, and yeast containing pGal1,10- IsPETase was the team’s experimental sample. The samples of the yeast, each containing two different composite parts, respectively, were induced in the presence of the galactose at 30’, 60’, 90’, and 120’, and the whole cell RNA extracted samples were operated before the RT-qPCR technique was performed.

The team conducted RT-qPCR to assess the induction of IsPETase mRNA levels downstream of the pGal1,10 promoter in yeast treated with 2% galactose. We used pGal1,10-eGFP as a control and measured eGFP mRNA induction at 0, 30, 60, 90, and 120 minutes. We observed a peak eGFP mRNA induction of approximately 8-fold at 120 minutes in the presence of 2% galactose compared to the 0-minute sample without galactose.

However, there was no significant induction of IsPETase mRNA across the 2-hour time course.

To further investigate the induction pattern of IsPETase mRNA, the team plans to collect additional time-course samples beyond the initial 2 hours of galactose treatment.

Protein Expression

To verify whether several different IsPETse mutants cloned downstream of the T7 promoter, were been manipulated to generate protein, the team set up the SDS page and western blot experiments, our team cultured 50ml of DE3BL21 bacteria cells containing T7-IsPETase and various mutation plasmids in LB broth medium until OD~0.2, respectively. Then cell culture was split into half (25mls) and induced at 0.5mM of IPTG at 37 degrees for 6 hours and the other half (25mls) as uninduced at 37 degrees for 6 hours as controls.

Our team cultured 50ml of DE3BL21 bacteria cells containing T7-IsPETase and the various mutated plasmids in LB broth medium until eac reached OD~0.2, respectively. The cell culture was then split into half (25mls) and induced at 0.5mM of IPTG at 37°C for 6 hours and the other half (25mls) was uninduced at 37°Cfor 6 hours as controls.

The team did whole cell protein extraction via the 95°C boiling method in protein extraction lysis buffer and ran samples on an an 18% SDS page gel to show a stronger band of the IsPETase along with various mutantions of IsPETase at the correct protein size in the presence of IPTG.

After conducting 18% SDS-PAGE gel analysis, our team proceeded with Western blotting to assess the expression of the IsPETase protein. We cloned the IsPETase gene downstream of a 6-his tag, which resulted in the expressed protein also having a his-tag at its N-terminal.

For the western blot, we utilized a his-tag primary antibody and an HRP-conjugated anti-mouse secondary antibody to detect IsPETase expression for various samples. However, we did not detect any distinct bands of the IsPETase protein expression, even in the presence of IPTG.

Moving forward, we need to optimize the Western blot conditions, as this procedure involves multiple steps and two antibody incubations. Additionally, the use of an incorrect antibody dilution may have contributed to the lack of strong IsPETase expression bands.

PET Degradation

The final more direct functional assay is to determine the efficiency of PET degradation through co-culture of the same amounts of PET film with either DE3BL21 bacteria containing T7-IsPETase plasmids along with various mutations on IsPETase enzyme, respectively in the presence of IPTG to induce T7 promoter in DE3BL21 as experimental samples, lack of IPTG as controls. The team used a Nabi machine, which detects 240 nm, to detect the amount of TPA product generated daily for the co-culture experiment. The same experiment was performed with yeast containing the pGal1,10-IsPETase plasmid, along with the pGal1, 10-eGFP as a control, in the presence of galactose as experimental samples, and in the glucose as controls.

After doing the PET co-culture functional assay, we did not find any pattern in the spectrometer results. Peaks were low (0.001-0.04 OD only) and we didn’t see the peaks appearing at the same wavelength (232-248 nm all had peaks). After discussing with our instructor, we concluded that these reasons may have caused it: First, our UV-visible spectrometer isn’t as accurate as the university ones, so the peaks might shift and cause the inconsistent wavelengths we observed. Secondly, the reason for the weak signal might be caused by too little products being made, so with Professor Ng’s suggestion, we decided to do a p-NPB assay first to ensure our plasmid does have activity, then use PET powder to co-culture to increase the surface area, therefore, increase the reaction rate (UCM team also suggested us if we couldn’t buy the powder, freeze our PET film then grind it, it would be easier). Professor Ng also suggested we use crude protein (whole cell extract) to do our experiments so the enzyme diffusing in and out of the cell wouldn’t cause another variable.

After closely analyzing the results, we also believe technical errors caused the inconsistent data. Every time a particular teammate collected the data, the absorbance was always higher. It was concluded that she shook the flasks more vigorously than the other teammates before taking the samples. To fix this problem, our team will keep the same member on all sample collections.

Citations

Brott, S., Pfaff, L., Schuricht, J., Schwarz, J., Böttcher, D., Christoffel, Wei, R., & Bornscheuer, U. T. (2021). Engineering and evaluation of thermostable IsPETase variants for PET degradation. Engineering in Life Sciences, 22(3-4), 192–203.

Maity, W., Maity, S., & Bera, S. (2021). Emerging Roles of PETase and MHETase in the Biodegradation of Plastic Wastes.

Rajeshkannan, Mahilkar, A., & Saini, S. (2022). GAL Regulon in the Yeast S. cerevisiae is Highly Evolvable via Acquisition in the Coding Regions of the Regulatory Elements of the Network. Frontiers in Molecular Biosciences, 9. https://doi.org/10.3389/fmolb.2022.801011

Sevilla, M. E., Garcia, M. D., Perez-Castillo, Y., Armijos-Jaramillo, V., Casado, S., Vizuete, K., Debut, A., & Cerda-Mejía, L. (2023). Degradation of PET Bottles by an Engineered Ideonella sakaiensis PETase. Polymers, 15(7), 1779.

Son, H. F., Cho, I. J., Joo, S., Seo, H., Sagong, H.-Y., Choi, S. Y., Lee, S. Y., & Kim, K.-J. (2019). Rational Protein Engineering of Thermo-Stable PETase from Ideonella sakaiensis for Highly Efficient PET Degradation. ACS Publications.

Yoshida, S., Hiraga, K., Taniguchi, I., & Oda, K. (2021). Ideonella sakaiensis, PETase, and MHETase: From identification of microbial PET degradation to enzyme characterization. Methods in Enzymology, 648, 187–205.