Abstract

PET plastic is one of the most commonly recycled plastics, often remade into various products. However, over the past few decades, the uncontrolled disposal of PET plastic waste worldwide has led to significant environmental damage and sparked widespread controversy. In response, industries and scientists are now focused on developing more cost-effective methods to break down PET plastic.

In order to increase the efficiency of recycling using biological enzymes (bio recycling), our research team concentrated on enhancing the activity of the PETase enzyme using site-directed mutagenesis, creating variant IsPETase enzymes. These IsPETase enzyme variants accelerate the breakdown of PET plastic into the monomers terephthalic acid (TPA) and mono-(2-hydroxyethyl) terephthalate (MHET). Our goal is for these IsPETase enzyme variants to be adopted at the industrial level to enable more efficient recycling of these monomers, therefore helping reduce plastic pollution.

The project could be extended to enhance a circular economy through further degrading the MHET into TPA and ethylene glycol (EG) with MHETase, a focus of our team’s future work on MHETase biobricks design (Kushwaha et al., 2023, Bertocchini & Arias, 2023).

Project Inspiration

Initial Inspiration

During a Biology course, we learned about endocrine disruption, a prevalent issue leading to cancer and chronic diseases, and one of its main causes, microplastics. In Taiwan, plastic bags are ubiquitous, and they are even used to hold steaming hot noodle soups from street vendors. These seemingly innocuous bags leak harmful chemicals, posing a threat to human health by misregulating the human body’s hormonal balance. Recognizing that plastic pollution is a global issue, our team decided to focus on mitigating the health risks posed by microplastics not only in Taiwan but also in many other countries where similar plastic usage practices are widespread.

Figure 1: Inspiration of our project

Problem

Plastics Pollution Status Quo

The global production of plastic has increased from 2 million metric tons in 1950 to 380 million metric tons in 2015 (Ritchie et al., 2023). Plastic gained popularity and widespread use primarily due to being lightweight, firm, affordable, versatile, hygienic, a good insulator, and easy to manufacture, offering significant advantages over traditional alternatives such as steel, paper, glass, and wood (Pilapitiya & Ratnayake, 2024). The post-World War II era and the 1960s and 1970s saw a surge in plastic production as consumers sought these benefits, leading to its adoption across various industries including medicine, furniture, transportation, packaging, and construction (Science History Institute, 2024). Today, that figure has increased to over 450 million tonnes annually (Parker, 2024). Despite their utility, plastics pose a grave environmental threat, particularly in their disposal and management (Parker, 2024).

According to the survey conducted by the Human Practice team, about 35.8% of respondents are unaware of the actual sources of microplastics and its effects. For instance, many were unaware that laundry is a source of microplastics and mistakenly believed that the food industry (44.4%) and chemical reactions (46.9%) were major contributors. Therefore, with the collaboration of wet lab and human practices, we aim to alleviate the detrimental effects of microplastics, specifically PET plastics.

Plastic's Impact on Human

The pervasive use of plastic has led to a series of detrimental effects on both ecosystems and human health (Pilapitiya & Ratnayake, 2024). As plastic waste accumulates, it traps and injures wildlife, including life on land and life below water, disrupting their habitats and causing a decrease in species populations (Pilapitiya & Ratnayake, 2024). The decrease in species population ultimately impacts the balance of the food chain, reducing biodiversity and habitat quality (Pilapitiya & Ratnayake, 2024).

Microplastics, which refer to small pieces of plastic less than 5 mm (0.2 inch) in length, occur as a consequence of plastic pollution (Rogers, 2024). In particular, microplastics inflict significant damage at the cellular level, resulting in the demise of numerous marine and terrestrial species (Rogers, 2024). Furthermore, the repercussions extend to human health, as exposure to plastic is linked to hormone imbalances leading to endocrine disruption, lung disease, insulin resistance, decreased reproductive health, and an increased risk of cancer (KOSOVO, 2023). Moreover, it is estimated that the average adult consumes approximately 121,000 microplastic particles per year (Horiba, n.d.). Microplastics and nanoplastics–smaller plastic particles less than one µm in size–can enter the human bloodstream and organs through inhalation, oral intake, skin contact, and direct consumptions such as bioaccumulative seafood and tap water (Rogers, 2024).

According to Harvard Health, heating plastics may cause chemicals or toxins to leak into food, leading annually to deaths between 400 thousand and 1 million (Harvard Public Health Review, 2019). Lastly, during interviews with Ms. Siña, we learned that there have been discoveries of microplastics in human vein or artery blockages, suggesting a potential correlation between microplastics and such blockages. This suggests that microplastics in the human body may have more severe consequences than we think, especially since plastics are now much more commonly used (M. Siña, personal communication, September 17, 2024).

Plastic’s Impact on Environment

Microplastics

What are PET plastics?

The resin identification code system divides plastics into seven categories by their structures and density, including polyethylene terephthalate (PET), high-density polyethylene (HDPE), polyvinyl chloride (PVC), low-density polyethylene (LDPE), polypropylene (PP), polystyrene (PS), and others (Plastics for Change, 2023). PET is one of the most common plastics we see in our daily lives. 390.7 million tons of plastic were produced, of which PET constituted 6.2% of plastic production. PET has a wide variety of uses from textiles to food packaging to plastic bottles (Plastics for Change, 2023).

Figure 6: Types of plastics with and without carbon backbone

Unlike HDPE, LDPE, PP, PS, and PVC, PET does not have a carbon backbone (Plastics for Change, 2023). PET is a polyester containing ethylene glycol and terephthalic acid as its repeating units and can be either semi-crystalline or amorphous depending on the conditions during crystallization. A quicker cooling process will result in a transparent amorphous structure. A slower cooling process produces a semi-crystalline structure, with a stronger, translucent product.

PET Degradation pathway

In recent decades, scientists have discovered several organisms, such as waxworms, bacteria, some protists, and fungi, can degrade plastics (Ruiz Barrionuevo et al., 2022; Palm et al., 2019; Rillig & Bonkowski, 2018; Ahmaditabatabaei et al., 2021). One of the bacterial strains, Ideonella sakaiensis, was discovered and well-studied with its PET hydrolase (PETase) and MHETase enzymes, which are essential for PET plastics degradation (Palm et al., 2019).

IsPETase degrades polyethylene terephthalate by hydrolyzing the ester bonds to form mono-(2- hydroxyethyl) terephthalate (MHET), bis-(2-hydroxyethyl) terephthalate (BHET), and terephthalic acid (TPA). The oligomers MHET and BHET could be later hydrolyzed into terephthalic acid (TPA) and ethylene glycol (EG) by a mutated MHETase as the team’s future goal (Khairul Anuar et al., 2022).

Figure 8: PET degradation pathway

Factors that can degrade plastics

Internal factors: Plastic biodegradability is influenced by various internal factors, which can be determined by examining certain physical and chemical properties. These include the availability of functional groups that increase hydrophobicity, and the complexity of the polymer structure, whether it is linear or branched (Pilapitiya & Ratnayake, 2024). The presence of easily breakable bonds, such as amide and ester bonds (with esters being more easily breakable than ethers, amides, or urethanes), and the type of coupling in the chain also play a role (Pilapitiya & Ratnayake, 2024). Additionally, the composition of the polymer at the molecular level, its form and physical appearance (e.g., pellets, films, or powder), and its density and molecular weight are important factors. The morphology, including the proportion of the amorphous and crystalline regions, as well as the tendency for soft polymers to degrade faster than hard polymers, further contribute to the biodegradability of plastics (Tokiwa et al., 2009).

External factors: Besides internal factors, external factors such as sunlight, heat, oxidation, or friction can break down plastic, but the process requires a long period, during which the fragments become hazardous (Pilapitiya & Ratnayake, 2024). The plastics can degrade through two different reactions: random chain scission and unzipping depolymerization (Liu et al., 2023). The former describes the long chains of the polymer randomly breaking into smaller pieces; the latter describes the polymer chains "unzipping", reverting back to their original monomer units. Going through these reactions allows plastics to break down (Tokiwa et al., 2009).

Taiwan’s Status Quo

Over the past ten years, Taiwan’s production of plastics and plastic-related industries have had a significant social and economic impact.

Economic Values:

• In 2020, the production value of plastic film in Taiwan reached $2.8 billion (Formosa Plastics Group at TITAS, 2021).
• The total annual production value of the global plastic and rubber machinery industry in 2012 was estimated at €29.2 billion. Taiwan ranked among the top 5 producers, along with Germany, China, Italy, and Japan (Taiwan Association of Machinery Industry, 2022).

Specifically regarding Taiwan's national efforts in implementing plastic recycling, in July 1991, the Taiwanese government restricted the use of plastic bags in the following 14 control subjects: (1) Public sector, (2) Private schools, (3) Department stores and shopping malls, (4) Mass retailers store industry, (5) Supermarket industry, (6) Chain convenience store industry, (7) Chain fast food restaurant public sector, (8) drug stores, beauty stores, and pharmacies, (9) medical equipment stores, (10) Retail of home appliances, photography, information, and communication equipment, (11) Retail of books and stationery, (12) Laundry Clothing store industry, (13) beverage store industry, (14) pastry bakery industry (Chang & Wu, 2024).

Efforts include:

• Taiwan's commitment to environmental sustainability has led to increased regulations on plastic production and usage by 2030. Also, the government has promoted recycling and the development of biodegradable alternatives by budgeting NT$200 million (US$6.14 million) for ESG (environmental, social, and governance) purposes, which can incentivize corporations to participate in the reduction of plastics and greenhouse gasses (Chang & Wu, 2024).
• Taiwan's annual use of plastic shopping bags has dropped from 20 billion in 2002 to 9 billion in 2023, a reduction of over 50%, indicating a significant decrease in plastic consumption (Chang & Wu, 2024).
• In 2019, Taiwan’s overall plastic recycling rate was 35%. The domestic recycling rate has increased steadily since 1997, reaching 51% in 2019 (Chang & Wu, 2024).

Taiwan’s transition to a circular economy is reshaping Taiwan's approach to plastics, with the government introducing strategies to enhance recycling and reduce waste, resulting in a domestic recycling rate of 51% as of 2019 (Wu et al., 2021). This transition not only addressed environmental concerns but also created economic opportunities in the recycling and green manufacturing sectors (Wu et al., 2021).

However, 91.44% of the plastic bags in Taiwan are used in traditional markets, snack stalls, and dine-in restaurants with storefronts, which are not included in the government-restricted subjects (Chang & Wu, 2024). The high usage in these policy-excluded groups highlights the ineffectiveness and lack of implementing the plastics-related regulation policy. According to the national report, the higher use of plastics in these places is due to convenience, potential for alternative use as garbage bags in the future, and Taiwanese citizens generally being unaccustomed to carrying eco-friendly bags (Chang & Wu, 2024).

Why Start from Recycling?

During one of the meetings with our school’s business teachers, they suggested we consider the root cause of widespread plastic pollution to maximize the impact of our solution. Therefore, among the various solutions to combat plastic pollution—such as clean-ups, biodegradable plastics, and others—we looked specifically into industry recycling methods. Improving recycling techniques for plastics is often prioritized over alternatives like clean-ups or biodegradable plastics due to major considerations such as economic feasibility, resource recovery, and regulatory support (Ritchie et al., 2023). Enhanced recycling methods also contribute to a circular economy by reusing plastics instead of discarding them (Hopewell et al., 2009). If we can find more economically efficient recycling methods, Georg Snyman, who works within the recycling industry, believed we could encourage its use, as the push factor of sustainability is not enough to encourage recycling in his opinion. This not only conserves resources but also results in higher-quality recycled materials, making them more valuable in the market and encouraging better waste management practices (Hopewell et al., 2009). Furthermore, improved recycling also allows for the recovery of valuable materials from waste streams, reducing the reliance on virgin plastic production and aligning with sustainability goals (Hopewell et al., 2009).

Pros and Cons of Traditional Recycling Techniques

To achieve sustainability and perseverance, PET recycling is a suitable method to mitigate the detrimental effects of plastic waste. PET recycling includes two commonly used techniques: mechanical recycling and chemical recycling (Joseph et al., 2024).

Figure 9: Current techniques for PET plastic recycling and their drawbacks

Mechanical Recycling

The first method, mechanical recycling, is the most common due to its cost-effectiveness and energy efficiency (Joseph et al., 2024). Mechanical recycling involves shredding PET plastic into smaller pieces, then cleaning, melting, and molding the pieces (Joseph et al., 2024). These steps separate the plastic structure from contaminants, melting them to create molten PET resin, and molds the PET resin into new products, such as bottles, fibers, or films (Joseph et al., 2024). However, there are two major drawbacks of mechanical recycling: reduced quality and negative environmental impact (Copper Sustainability Partnership, 2024). First, mechanical recycling involves repeated processing of plastics, leading to degradation of the material (Copper Sustainability Partnership, 2024). This results in the quality of recycled plastic possibly being lower than the original, with this lower quality plastic eventually being discarded in landfills, highlighting the inefficiency of this method. The process of melting down and recycling plastic also produces volatile organic compounds (VOCs), harming plants and animals near the industrial site (Cabanes et al., 2020). Additionally, the heat required to melt plastic generates carbon emissions, contributing to global warming (Copper Sustainability Partnership, 2024).

Chemical Recycling

The second method, chemical recycling, involves chemical reactions such as glycolysis and methanolysis to break down the extended chains of PET molecules into individual high-quality monomers, utilizing these monomers to produce new PET products (Bohre et al., 2023). The procedure encompasses various stages, including depolymerization, purification, re-polymerization, and forming. Chemical recycling of PET presents numerous advantages compared to mechanical recycling, including enhanced resilience and versatility in the resulting product (Bergamin, 2024). Moreover, it can generate high-quality recycled PET with properties closely resembling virgin PET, rendering it suitable for demanding applications such as food packaging and automotive components(Joseph et al., 2024). Nevertheless, the significant obstacles of chemical recycling lie in scalability and commercial viability, underscoring its inability to replace the mechanical recycling method (Bergamin, 2024). First, chemical recycling is still in the early stages of development, and this technical challenge limits the facilities to conduct small-scale tests and practices. Furthermore, even though chemical recycling has the potential to produce high-quality recycled plastics that are indistinguishable from new plastic, the quality varies depending on the specific process used, demonstrating the challenge of producing consistent high-quality recycled plastic (Zhang, 2023). According to the Environmental Health News Reports, most outputs of chemical recycling are combustible fuels or chemicals that do not alleviate the demand for virgin plastics (Zhang, 2023).

All in all, these traditional methods can reduce the amount of PET products that end up in landfills or oceans, and contribute to developing a circular economy. However, each method has limitations, such as the quality and quantity of collected PET products, contamination, and material quality. Thus, our team looked into a third growing option for plastic recycling.

Bio-recycling

Bio-recycling, also known as enzymatic recycling, is an innovative approach utilizing enzymes to break down PET products into their monomers, involving 5 distinct steps for PET degradation: collection and sorting of PET waste, size reduction, enzymatic depolymerization, purification, and polymerization (U.S. Government Accountability Office, 2022). This method currently stands as the most environmentally friendly approach, mitigating the impact of plastic waste without relying on fossil fuels, even for processing low-quality PET waste. Additionally, it yields high-quality monomers suitable for producing new PET with properties akin to virgin PET.

Furthermore, bio-recycling has the potential to decrease greenhouse gas emissions, reduce energy consumption, and enhance the overall efficiency of PET plastic degradation product recycling. Despite these promising aspects, recycling is in its early stages and requires further research and optimization for commercial viability, posing challenges such as scalability and environmental impacts, while also being time-consuming. Specifically, scalability remains as the biggest limitation of bio-recycling as it is a relatively new technique in its early experimental stage, making it difficult to scale up operations to meet industrial demands (Joseph et al., 2024). Moreover, challenges like limited applicability and implementation costs are all factors to be considered when introducing this relatively new recycling technique (U.S. Government Accountability Office, 2022).

After evaluating the advantages and importance of recycling in the status quo, our team decided to work on the bio-recycling technique, as it could tackle the issue most effectively and efficiently due to its environmentally friendly nature and potential for creating monomers that can be returned into the plastic production cycle. After considering the limitations of bio-recycling, our team aimed to devise a solution that improves the quantity and quality of the PETase enzyme activity.

SDG Connection

For guidance on our project's potential impacts and focus, we looked to the UNSDGs. Our project aligns with four of the 17 goals — #3: Good Health and Well-Being, #6: Clean Water and Sanitation, #12: Responsible Consumption and Production, and #14: Life Below Water.

Target 3.9 of Goal 3 aims to reduce illness from health hazards caused by chemicals in the air, water, and soil. Our project, which focuses on controlling the release of microplastics and PET into the environment by increasing recycling efficiency, helps prevent said hazards.

Our project also lines up with Target 6.3 of Goal 6, which focuses on minimizing the release of chemicals into water sources. By increasing PET degradation rates, we aim to increase recycling and decrease dumping, effectively reducing the release of PET waste and microplastics into bodies of water.

Goal 12 aims for a sustainable cycle of consumption and production. As bio-recycling does not release toxins into the air, water, and soil (the focus of Target 12.4), we are providing a method for companies to recycle using more sustainable methods.

Lastly, Goal 14 focuses on aquatic life, with indicator 14.1.1 specifically focusing on plastic debris density in the ocean. Again, by increasing recycling and decreasing dumping, our project provides an effective method to control the amounts of plastic entering the water.

Design

Inspiration

Our team checked previous iGEM teams' projects related to PET plastic degradation, and “Making bacteria eat plastic" by the 2018 Yale University team got the team’s attention (Yale iGEM). The Yale iGEM team used the PETase gene for their project (Yale iGEM, 2018), and our team wanted to extend their project by making an enhanced PETase enzyme with 3 amino acid mutations utilizing a strain of Ideonella sakaiensis 201-F6, IsPetase^{S121E/D186H/R280A}.

The team also upgraded the original Yale project, which used bacteria for induction only. Our team expressed the cloned IsPETase, along with several mutations on the IsPETase gene respectively, downstream of the pGal 1,10 promoter for the yeast strain, Saccharomyces cerevisiae, which can induce 20-30-fold induction in the presence of galactose.

Furthermore, the 2019 University of Toronto team performed mutations in the IsPETase gene to computationally optimize the enzyme’s thermostability and catalytic efficiency (University of Toronto iGEM, 2019). With a slightly different approach, our team focused more on the approach of bioengineering, but with the same goal of enhancing catalytic efficiency through the inclusion of mutations. Their project highlighted that the breakdown products of PET—terephthalic acid (TPA) and ethylene glycol (EG)—can have valuable applications (University of Toronto iGEM, 2019). Inspired by this, our team made it a priority to track TPA concentrations to evaluate the effectiveness of our mutated PETase enzyme.

Gene and Protocols Introduction

IsPETase

PETase is an enzyme that catalyzes the breakdown of polyethylene terephthalate (PET) plastic through hydrolysis, resulting in the formation of MHET, BHET, and terephthalic acid (TPA). The team will use a strain of bacteria called Ideonella sakaiensis 201-F6, found in sludge samples collected near a PET bottle recycling site in Japan (Yoshida et al., 2016). Studies by Japanese groups showed that IsPetase has more tolerance for catalytic activities to degrade PET plastic in variable environments (Yoshida et al., 2016).

IsPETase has been confirmed to have the highest PET degradation activity of all PET-degrading enzymes from different microorganisms, as evidenced by its structure and chemical bonding (Anuar, 2022). Furthermore, IsPETase possesses two disulfide bonds in its active site, contributing to its efficiency in PET substrate binding. After many studies and experiments, IsPETase was found to be a promising candidate for PET degradation at both ideal temperatures and room temperature. Therefore, the team chose IsPETase to synthetically biodegrade PET plastic.

T7 Promoter Plasmid in DE3 Bacteria

The T7 promoter is a sequence of DNA used to regulate the gene expression of recombinant proteins in various applications (Tabor, 2021). The T7 RNA polymerase specifically recognizes T7 promoter in the bacterial competent cells of BL21(DE3)pLys. This allows high efficiency of RNA transcription of the gene cloned downstream of the T7 promoter when the promoter is induced by IPTG. also has a ribosome binding site for high-efficiency protein expression. The T7 promoter plasmid also contains an N-terminal 34aa precursor peptide with a histidine tag sequence, 6-his tag, to bind to the nickel-resin column for the in vitro protein purification to extract the proteins our team is interested in (Merck, n.d.). The ampicillin-resistant gene is also synthetic and cloned into the T7 promoter plasmid for bacterial transformation selection on LB-Amp plates. The recognition and resulting high efficiency of RNA transcription made the T7 promoter plasmid the suitable candidate for our experiment.

pGal1,10 Promoter Plasmid in Yeast

The pGal1,10 plasmid can be switched on in the presence of galactose and switched off in the presence of glucose in the medium. The benefit of using the pGal1,10 promoter is to regulate the cloned gene’s expression by adding either glucose or galactose into the yeast medium to turn off or on the gene transcription (Connor, n.d.). Our team will adopt this pGal promoter mainly due to its high expression (20-30 fold induction) of the genes related to this project cloned downstream of the pGal promoter in the presence of galactose for the in vivo protein expression in yeast.

Our team will clone the IsPETase gene downstream of the T7 promoter in DE3 bacteria, and downstream of the pGal 1,10 promoter in yeast. Our team will then perform site-directed mutagenesis to create a genetically engineered mutant IsPETase that is capable of degrading PET plastic more efficiently, as well as more compatible to function at a wider range of conditions, consequently reducing the amount of plastics and microplastics that are in the ocean.

Our approach

Our team aims to enhance PETase enzyme activity by creating three specific amino acid mutations on the PETase enzyme (IsPETase^{S121E /D186H/R280A}) under inducible promoters, T7, and pGal1,10.

Experimental Steps

Making IsPETase plasmids

After receiving the synthesized IsPETase gene from Mission Biotech, our team designed the forward and reverse primers flanked respectively with BamHI and HindIII double enzymes. We then did PCR to amplify the gene. After amplification, we performed double enzyme digestion using our double enzymes on the PCR product and the T7 promoter plasmid, which generates compatible sticky ends on the PCR product and the plasmid, which allowed us to further perform T4 ligation to ligate the digested IsPetase gene to the T7 promoter plasmid. We then transformed the T7-IsPetase into bacteria and did LB-amp plate selection and bacterial inoculation, which will select and amplify the bacteria containing our cloned plasmid. After the inoculation, we extracted our plasmid, and performed PCR again to confirm our cloning success.

For the yeast, the procedure was similar to that of making T7-IsPETase, except for the double enzymes used for double enzyme digestion, which were XmaI and SpeI, instead of BamHI and HindIII. For the rest of the procedure, the making of pGal 1,10-IsPetase was identical to that of the experiments done when making T7-IsPETase.

Site-Directed Mutagenesis

To design mutations on the IsPETase gene, our team designed point mutations on the forward and reverse primers, designing S121E/D186H/R280A single, double, and triple mutations. Then, we performed PCR amplification with these mutated primers to amplify the mutant plasmid. As we only wanted the mutated plasmid, we did DpnI digestion to digest the parental plasmid containing methylation markers, leaving only the mutant plasmid. After we obtained the mutated plasmid, we again transformed it into bacteria and did LB-amp plate selection and inoculation to select and amplify the bacteria containing our plasmid. We then extracted the plasmid from the bacteria and sent the sequences to Mission Biotech to check our mutation results.

Functional Assays

RT-qPCR

For our first functional assay, we did RT-qPCR to check for mRNA expression in our yeast. We first did a time course sample selection: after growing the engineered yeast in glucose, we transferred each engineered yeast into a galactose medium and then collected time course samples in 30-minute time stamps. We then performed a whole-cell RNA extraction and did an RT-qPCR experiment.

Western blot

To verify protein production in bacteria, we also did western blot experiments to check for protein expression. Before setting up the SDS page and western blot experiments, we cultured each engineered bacteria in LB broth medium. Each sample of engineered bacteria was separated into a flask containing IPTG, and another without IPTG as control. Then, for each sample, we did a whole cell protein extract of those bacteria, then performed SDS page and western blot experiments to verify protein expression.

Co-Culture Experiments

Lastly, we designed a co-culture experiment to verify plastic degradation, where we checked for TPA, a product of PET plastic after plastic degradation. For bacteria, we cultured each sample containing our mutated plasmids, with the same amount of PET film, in a flask containing IPTG to induce the T7 promoter, and in another flask not containing IPTG as control. For our yeast, we cultured each sample containing our mutated plasmids, with the same amount of PET film, in a flask containing galactose medium as the experimental group, and a flask containing glucose as control. We then used a Nabi machine with 240 nm wavelength to detect the amount of TPA.

Lab Extensions

Besides in vivo experiments, our team can do in vitro protein purification to extract the IsPETase enzyme and each of the several amino acid mutations of the IsPETase enzymes, respectively. The downstream of this T7 promoter is engineered to contain a 6 histidine-tag (6-his tag). Once we’ve successfully cloned the IsPETase gene to the downstream of the 6-his tag, along with each of the respective amino acid mutations of the IsPETase enzymes, the team will transform them into BL21DE3 bacteria to express those enzymes in the presence of IPTG.

After transforming those wild-type and different mutations of the IsPETase enzymes with 6-his tag on into DE3BL21, bacteria will be induced in the presence of IPTG for 6 hours to express each of the respective enzymes. Then the team will perform whole-cell protein extraction with the lysis buffer and run the whole-cell extract samples through the Ni+-resin column, which has an affinity with the 6-his tag. The wild-type IsPETase and several mutations of the IsPETase containing 6-his tag will be purified through the Ni+resin column after several chemical buffers are used to wash through the column to eliminate unbound proteins.

Application

Specialists’ Suggested Future Directions

Scientist Howard Cheng recommended that biodegradation be carried out in factories, where temperature, toxicity, and other factors can be controlled, to avoid causing biological toxicity and environmental harm.

Mr George Snyman, who works on encouraging the adoption of recycling, emphasized that rather than focusing solely on individual awareness for small-scale recycling, it's more effective to engage companies, both local and larger corporations. He noted that many large companies have corporate social integration (CSI) funds that can support small businesses and projects with detailed plans. Using Coca-Cola as a successful example, he suggested that a clear proposal with detailed, supporting lab results could attract funding for industrializing our project. Furthermore, Mr. Snyman indicated that while governments make changes in law typically requiring 3~5 years of effort, the increasing focus on plastic pollution and environmental issues may expedite this process, presenting an opportunity to advance our project with a solid business plan and ongoing successful experimentation.

Implementation of Our Final Product

Our method of pursuing bio recycling through PETase enzymes is not the first of its kind. In fact, Carbios, a French-based company, uses enzymatic PET recycling on an industrial scale. This means that our product, in the form of mutated PETase enzymes, does not require extra alterations to be directly used on the industrial scale. Furthermore, with the possibilities of partnership we learned from Mr. Snyman, we can outline the future expectations of our entire product. First, after completing purification and achieving a higher degradation rate in the lab, we can provide a detailed plan on how our product could be implemented.

Second, using the detailed plan, we could reach out to local or smaller environmental groups and form partnerships, which then help us use our product on a small firm scale. Thirdly, as Mr. Snyman suggested, through an executable plan and collaborative partnerships with other organizations, applications for CSI funding in large-scale products could then help increase production sizes. Lastly, if possible, the long-term plan is to apply for government law enforcement, which, although time-consuming, would ensure the implementation of more sustainable methods by law.

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