Introduction

The creation of Biobricks via cloning is central to synthetic biology research and a fundamental technique for iGEM projects. Recognizing the importance of synthetic biology skills, especially during the COVID-19 pandemic, Kang Chiao International School (KCIS) in New Taipei, Taiwan, established the Synthetic Bioresearch Lab in September 2021. The current 2023-24 team is the third iGEM team from KCIS Xiugang. From September to December 2023, all team members learned various genetic modification processes and techniques, including PCR, designing restriction sites on primers, NEB double enzyme digestion, T4 ligation, bacterial transformation, and site-directed mutagenesis as part of their bench work. They also developed bioinformatics skills, utilizing tools like NCBI to access publications and gene sequences. After the first semester of learning cloning techniques, the class was separated into 3 groups, and each came up with a topic for the project and then presented their idea to the others. The voting of the final decision was done in mid-December of 2023 and our team could spend time researching more publications to come out with the entire project’s outline during the winter break, late January of 2024. This year, the team’s inspiration came from a Biology course when the teacher mentioned endocrine(hormones) misregulation, which would cause cancer and chronic disorders. In Taiwan, using plastic bags is very common everywhere, even if you purchase hot noodle soups, the owners of the food stand still use plastic bags for them. Plastic bags release chemical materials harmful to the human body, even misregulating the hormones. 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 (Team:Yale, 2018). The Yale iGEM team also used the PETase gene for this project. Our team upgraded the PET plastic degradation project to make an enhanced PETase enzyme with several amino acid mutations from a strain of bacteria called Ideonella sakaiensis 201-F6, cloned downstream of either the T7 promoter for the DE3 bacteria or the pGal1, 10 promoter for the yeast.ational School (KCIS), in Taipei, Taiwan, the school realised the importance of synthetic biology skills during the COVID-19 pandemic. As a result, the school fully supported Ms. Parker and Ms. Kuo when they proposed setting up a Synthetic Biology lab, starting in September 2021. Ms. Kuo had previous experience training those in the medical field on how to complete cloning techniques. Students attended a class every Thursday from periods 5 to 8 to learn the basic protocols involved with cloning. They then applied what they had learnt in the class to the iGEM competition.

Our Final Goal and Design

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 biodegradable methods to break down PET plastic. Our research team concentrated on enhancing the activity of the PETase enzyme using site-directed mutagenesis, creating the 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 help reduce plastic pollution and enable more efficient recycling of these monomers. The MHET will then be further degraded into TPA and ethylene glycol (EG) by an MHETase, a focus of our team’s future work on MHETase biobricks design (Anamika et al, 2023; Bertocchini et al, 2023).

Several Amino Acid Mutations In The IsPETase Gene The Team Created

Several publications did further investigations 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 at the low PH and high-temperature environment to degrade PET plastic degradation (Brett et al, 2021; Maity et al, 2021; Son et al, 2019).

Our team aimed to enhance PETase enzyme activity by creating three specific amino acid mutations on the PETase enzyme (IsPETase^{S121E/D186H/R280A}) under two inducible promoters, T7, and pGal1,10, to express as many as the team desires. This approach would significantly increase PETase efficiency and allow catalytic activity in a fluctuating environment, thereby accelerating PET plastic degradation. Currently, industry applications rely on the wild-type IsPETase for PET degradation. Our goal is to develop and commercialize these enhanced PETase variants to substantially improve plastic degradation efficiency.

However, the IsPETase gene sequence contained multiple DNA fragments with repeat regions that our team didn’t expect while designing those mutations, leading to challenges for the site-directed mutagenesis technique. The primers containing mutant nucleotides that our team desired to switch specific amino acids targeted incorrect amino acids due to these repeats so our team created T7-IsPETase, T7-IsPETase^Thr116Ala, T7-IsPETase^{Thr116Ala/Lys259Glu}, T7-IsPETase^{Thr116Ala/Met154Thr} instead. For the pGal1,10 promoter, our team couldn’t make any mutations on the IsPETase gene, so the team has pGal1,10-IsPETase, and pGal1,10-eGFP utilized as the control (BBa_K418008) created by 2022 KCIS team. The team still decided to use those IsPETase mutations downstream of the T7 promoter for further experiments because other publications showed altered different amino acids in the IsPETase gene, to enhance the enzyme activity, our team hypothesized the mutations created by accident might also affect the catalytic activity (Sevilla et al, 2023).

Basic Parts

The first two basic parts our team needed were two inducible promoters that could be easily switched on/off for the downstream cloned gene’s RNA transcription. BBa_K5094000:T7 promoter on pRSET A plasmid was a type of common prokaryotic expression vector, designed for high-level prokaryotic expression. The downstream T7 promoter incorporated one DNA sequence, which encoded a 6-his tag, to bind to the nickel-resin column for the in vitro protein purification. T7 promoter was manipulated in the presence of isopropyl β-Dthiogalactoside (IPTG) to trigger our composition parts for protein expression. Our team also expressed the mutants of IsPETase along with the wild-type IsPETase, respectively, for the PET-degradation activity comparison. For future experiments, the team will extract the IsPETase enzyme and several IsPETase mutant enzymes via in vitro protein purification for in vitro enzyme-substrate assay.

BBa_K5094001: Gal1, 10 promoter was an inducible yeast promoter. Scientists engineered yeast Gal1, and Gal10 promoters into one Gal1, 10 promoter on the plasmid for synthetic cloning used as a switch-on in the presence of galactose, and switch-off in the presence of glucose in the medium (O’Connor, 2014). The benefit of using the pGal1,10 promoter was to regulate the cloned gene’s expression highly efficiently by adding either galactose or glucose into the yeast medium to turn on or off the gene transcription (Rajeskannan et al, 2022). Our team will manipulate the pGal1, 10 promoter for the in vivo functional assay to determine if these several IsPETase mutant enzymes show stronger PET-degradation levels than the IsPETase.

The IsPETase gene sequence suppressed our team with unexpected challenges during the site-directed mutagenesis technique due to multiple 5-10 nucleotide repeat regions that we hadn’t anticipated when designing the mutations. The primers, intended to switch specific amino acids by adding mutations to the primers, targeting incorrect amino acids because of these repeats. As a result, our team created alternative IsPETase mutant constructs: T7-IsPETase as a wild-type, T7-IsPETase^Thr116Ala, T7-IsPETase^{Thr116Ala/Met154Thr}, and T7-IsPETase^{Thr116Ala/Lys259Glu}. Despite these setbacks, our team decided to use the IsPETase mutations downstream of the T7 promoter for further experiments. This decision was based on previous publications demonstrating how altering several amino acids in the IsPETase gene could enhance enzyme activity. We hypothesize that the unexpected mutations the team created might also enhance the enzyme’s catalytic activity (Sevilla et al, 2023).

BBa_K5094002: IsPETase was the wild type of I. Sakaiensis bacteria gene. Before creating our team’s enhanced mutants of the IsPETase, the wild type of PETase gene was cloned downstream of either the T7 promoter or the pGal1,10 promoter first via traditional enzyme digestion cloning technique, to generate two composite parts ( BBa_K5094006 and BBa_K5094010). Those two composite parts were used as DNA templates for the site-directed mutagenesis to make several mutations of IsPETase. BBa_K5094006 and BBa_K5094010 composite parts were also the controls for functional assays

BBa_K5094003: IsPETase^Thr116Ala was a single mutation on the amino acid 116 in the IsPETase gene. The amino acid 116 from the polar uncharged side chain of the threonine switched to the hydrophobic side chain of the alanine, which would change the properties of the 116 amino acid, such as the interaction with the substrate or the catalytic function.

BBa_K5094004: IsPETase^{Thr116Ala/Met154Thr} was a double mutation on the amino acids 116 and 154 in the IsPETase gene. The amino acid 116 from the polar uncharged side chain of the threonine switched to the hydrophobic side chain of the alanine, and the amino acid 154 from the hydrophobic side chain of the methionine to the polar uncharged side chain of the threonine, which would change the properties of the 116 and 154 amino acids, such as the interaction with the substrate or the catalytic function.

BBa_K5094005: IsPETase^{Thr116Ala/Lys259Glu} was a double mutation on the amino acids 116 and 259 in the IsPETase gene, The amino acid 116 from the polar uncharged side chain of the threonine switched to the hydrophobic side chain of the alanine, and the amino acid 259 from the positive charge of the side chain on lysine to the negative charge of the side chain on the glutamic acid, which would change the properties of the 116 and 259 amino acids, such as the interaction with the substrate or the catalytic function.

Composite Parts

BBa_K5094006: T7-IsPETase (BBa_K5094000-BBa_K5094002): After amplifying the wild type of IsPETase gene via PCR and using enzyme digestion strategy to clone it to the downstream of the T7 promoter to make a composite part, BBa_K5094006, used to make several mutations on IsPETase gene via site-directed mutagenesis, and was transformed into DE3BL21 bacteria for functional assays.

BBa_K5094007: T7-IsPETase^Thr116Ala (BBa_K5094000-BBa_K5094003): After using BBa_K5094006 as a DNA template to do site-directed mutagenesis and the team designed the first mutation on the forward and the reverse primers, originally switching amino acid 121 from serine to glutamic acid (S121E). PCR was performed, and the parental DNA template was digested by DpnI and transformed into bacteria. However, the IsPETase gene sequence included multiple 5-10 nucleotide repeat regions that our team didn’t anticipate during the design of the mutations, which led to challenges in using the site-directed mutagenesis technique. The primers, which were intended to switch specific amino acids by introducing mutant nucleotides, ended up targeting incorrect amino acids due to these repeat regions. Our team created T7-IsPETase^Thr116Ala instead.

Fig1: IsPETase^Thr116Ala mutation involves a change at amino acid 116, where the polar uncharged threonine is replaced by the hydrophobic alanine. This alteration modifies the properties of amino acid 116, potentially affecting substrate interactions and altering the enzyme's catalytic function.

BBa_K5094008: T7-IsPETase^{Thr116Ala/Met154Thr} (BBa_K5094000-BBa_K5094004): After using BBa_K5094007, as a DNA template to do site-directed mutagenesis and the team designed the 2nd mutation on the forward and the reverse primers, originally switching amino acid 186 from aspartic acid to histidine (D186H). PCR was performed, and the parental DNA template was digested by DpnI and transformed into bacteria. However, the IsPETase gene sequence included multiple 5-10 nucleotide repeat regions that our team didn’t anticipate during the design of the mutations, which led to challenges in using the site-directed mutagenesis technique. The primers, which were intended to switch specific amino acids by introducing mutant nucleotides, ended up targeting incorrect amino acids due to these repeat regions. Our team created T7-IsPETase^{Thr116Ala/Met154Thr} instead.

Fig2: IsPETase^{Thr116Ala/Met154Thr} involves double mutations at amino acids 116 and 154 in the IsPETase gene. In this modification, the polar uncharged threonine at position 116 is replaced by the hydrophobic alanine, while the hydrophobic methionine at position 154 is substituted with the polar uncharged threonine. These changes in amino acid properties may alter substrate interactions and potentially impact the enzyme's catalytic function.

BBa_K5094009: T7-IsPETase^{Thr116Ala/Lys259Glu}. (BBa_K5094000-BBa_K5094005): After using BBa_K5094007, as a DNA template to do site-directed mutagenesis, the team designed the mutation on the forward and the reverse primers, originally switching amino acid 280 from arginine to alanine (R280A). PCR was performed, and the parental DNA template was digested by DpnI and transformed into bacteria. However, the IsPETase gene sequence included multiple multiple 5-10 nucleotide repeat regions that our team didn’t anticipate during the design of the mutations, which led to challenges in using the site-directed mutagenesis technique. The primers, which were intended to switch specific amino acids by introducing mutant nucleotides, ended up targeting incorrect amino acids due to these repeat regions. Our team created T7-IsPETase^{Thr116Ala/Lys259Glu} instead.

Fig3: IsPETase^{Thr116Ala/Lys259Glu} involves double mutations at amino acids 116 and 259 in the IsPETase gene. In this modification, the polar uncharged threonine at position 116 is replaced by the hydrophobic alanine, while the positively charged lysine at position 259 is substituted with the negatively charged glutamic acid. These changes in amino acid properties may alter substrate interactions and potentially impact the enzyme's catalytic function.

BBa_K5094010: pGal1, 10-IsPETase (BBa_K5094001-BBa_K5094002): After amplifying the wild type of IsPETase gene via PCR and using enzyme digestion strategy to clone it to the downstream of the pGal1,10 promoter to make a composite part, BBa_K5094010 was used to make several mutations on IsPETase gene via site-directed mutagenesis, and was transformed into a yeast strain, Saccharomyces, for functional assays. The team used BBa_K5094010 as a DNA template to make mutations but failed due to the large size of the pGal1,10-IsPETase plasmid in the ~7kb plasmid. The team will further optimize several primer sets of annealing conditions for the site-directed mutagenesis.

Part Tests

PCR and Electrophoresis

Our team did PCR techniques and ran PCR products on gels. The PCR products on the gels showed evidence of proof of concept.

Fig 4: BBa_K5094002, IsPETase full length product, 873bp

Fig 5: After bacterial transformation for T7-IsPETase cloning, bacteria colonies picked up directly to do IsPETase PCR.3 colonies on lane 2,3,4 had BBa_K5094006 Composite part (full-length IsPETase PCR product).

Fig 6: BBa_K5094002 IsPETase full-length product, 873bp

Fig 7: After bacterial transformation for pGal-IIsPETase cloning, bacteria colonies were picked up directly to do IsPETase PCR. Lane 2 was IsPETase PCR product control. 2 colonies on lanes 5 and 6 had BBa_K5094010 composite part (full-length IsPETase PCR product)

Electrophohresis and Sequencing

The team also ran those variants of the IsPETase mutants cloned downstream of the T7 promoter or the pGal1,10 promoter on the agarose gel showing evidence of the proof of the concept.

Fig 8: After sequencing, the mutations of the IsPETase the team created: lane 1 was a 1kb DNA ladder, and lane 2 was T7-IsPETase, lane 3 was T7-IsPETase^Thr116Ala, lane 4 was T7-IsPETase^{Thr116Ala/Met154Thr}, and lane 5 was T7-IsPETase^{Thr116Ala/Lys259Glu}

Fig 9: After sequencing, the team only created pGal1,10-IsPETase plasmid (lanes2, 3, 4), and lane1 is a 1kb DNA ladder.

Sequence Data

The IsPETase gene sequence gave our team unexpected challenges during the site-directed mutagenesis technique due to multiple DNA fragments containing repeat regions that we hadn’t anticipated when designing the mutations. The primers, intended to switch specific amino acids by adding mutations to the primers, targeting incorrect amino acids because of these repeats. As a result, our team created alternative IsPETase mutant constructs: T7-IsPETase as a wild-type (BBa_K5094006), T7-IsPETase^Thr116Ala (BBa_K5094007), T7-IsPETase^{Thr116Ala/Met154Thr} (BBa_K5094008), and T7-IsPETase^{Thr116Ala/Lys259Glu} (BBa_K5094009) for DE3BL21 bacteria. pGal1,10-IsPETase (BBa_K5094010) for the yeast. Despite these setbacks, our team decided to use the IsPETase mutations downstream of the T7 promoter for further experiments. This decision was based on previous publications demonstrating how altering several amino acids in the IsPETase gene could enhance enzyme activity. We hypothesize that the unexpected mutations the team created might also enhance the enzyme’s catalytic activity.

Fig 10: T7-IsPETase (BBa_K5094006) plasmid map after sequencing.

Fig 11: T7-IsPETase^Thr116Ala (BBa_K5094007) plasmid map after sequencing.

Fig 12: T7-IsPETase^{Thr116Ala/M154Thr} (BBa_K5094008) plasmid map after sequencing.

Fig 13: T7-IsPETase^{Thr116Ala/K259Glu} (BBa_K5094009) plasmid map after sequencing.

Fig 14: pGal1,10-IsPETase (BBa_K5094010) plasmid map after sequencing.

RT-qPCR was Conducted to Demonstrate that the Cloned Genes Downstream of the pGal1, 10 Promoter Were Induced in the Presence of Galactose

To manipulate our team’s wild-type IsPETase enzyme and the control eGFP, both cloned downstream of the pGal1,10 promoter, we used BBa_K418008, which contained pGal1,10-eGFP as the control, and BBa_K5094010, which contained pGal1,10-IsPETase as the experimental sample. These constructs were then transformed into wild-type Saccharomyces Cerevisiae yeast strain, BY4741, for further experimentation. After collecting different time course samples, the whole cell RNA extracted was operated before the RT-qPCR technique was performed.

Fig 15a: The mRNA induction of eGFP showed strong manipulation, above 5-fold induction, in the presence of galactose at 60 mins, and the maximum of 7.5-fold eGFP mRNA induction at 120 mins in the presence of galactose, which indicated 2% galactose was sufficient to trigger mRNA induction.

Fig 15b: The mRNA induction of IsPETase didn’t exhibit significant induction at different time courses in the presence of galactose compared to the 0’ sample with glucose only. To further determine an IsPETase mRNA induction timecourse pattern, the team will collect multiple time-course samples in the presence of the galactose behind 2 hours.

SDS Gel Electrophoresis

Before setting 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 (Mühlmann et al, 2017; Namdev et al, 2019).

To verify whether those composite parts, BBa_K5094006 containing BBa_K5094000- BBa_K5094002 (T7-IsPETase), BBa_K5094007 containing BBa_K5094000- BBa_K5094003 (T7-IsPETase^Thr116Ala), and BBa_K5094008 containing BBa_K5094000- BBa_K5094004 (T7-IsPETase^{Thr116Ala/Met154Thr}), and BBa_K5094009 containing BBa_K5094000-BBa_K5094005 (T7-IIsPETase^{Thr116Ala/Lys259Glu}), can express the proteins our team desires, the team did the whole cell protein extract via the 95-degree boiling method in protein extract lysis buffer protocol, according to the data shown on BBa_K2010000 from the Harvard 2017 team, the IsPETase protein was in the soluble condition, and the supernatants will be collected for the SDS page and Western blot. The team performed an 18% SDS page first. To confirm whether the whole cell extract contained all proteins and also see a stronger band of the IsPETase along with various mutants of IsPETase at the correct protein size in the presence of IPTG, an 18% SDS page image was exhibited in Fig 16.

Fig 16: The 18% SDS-PAGE gel showed the team successfully extracted the whole protectin extract. Lane 1 is a protein-size ladder. Lane 2 is T7-IsPETase (-)IPTG,and lane 3 is T7-IsPETase (+0.5mM)IPTG. Lane 4 is T7-IsPETase^Thr116Ala (-)IPTG, and lane 5 is T7-IsPETase^Thr116Ala (+0.5mM)IPTG. Lane 6 is T7-IsPETase^{Thr116Ala/Lys259Glu} (-)IPTG, and lane 7 is T7-IsPETase^{Thr116Ala/Lys259Glu} (+0.5mM)IPTG. Lane 8 is T7-IsPETase^{Thr116Ala/Met154Thr} (-)IPTG, and lane 9 is T7-IsPETase^{Thr116Ala/Met154Thr} (+0.5mM)IPTG. IsPETase protein has about 30 kDa molecular weight based on the Harvard 2016 team’s part information.

Co-culture with PET film experiments

The more direct functional essay was to co-culture the same amount/size of PET film with either DE3BL21 bacteria containing T7-IsPETase plasmid along with various mutations on IsPETase enzyme, respectively in the presence of 0.5mM IPTG to induce T7 promoter in DE3BL21 at 37 degrees as experimental samples, lack of IPTG as controls. The team used a Nabi machine with a “ 240 nm” wavelength to detect the total TPA product generated after the PET plastic degradation daily at 9 am and 3 pm for the co-culture experiment (Terephthalic acid, 2023). The same experiment was performed with BY4741 yeast strain containing the pGal1,10-IsPETase plasmid as an experimental sample, along with pGal1,10-eGFP as a control sample in the presence of galactose, samples in the glucose as comparison group without inducing the pGal1,10 promoter.

Fig 17a: The co-cultured experiment data didn’t show any clear patterns of the TPA product increasing in the DE3 bacteria containing T7-IsPETase along with several IsPETase mutants, respectively, in the presence of the IPTG during the time course experiment. For the experimental controls, DE3 bacteria only, along with several IsPETase mutants that lack IPTG still also showed TPA product generation.

Fig 17b: The co-cultured experiment data didn’t show a clear pattern of the TPA product increasing in the BY4741 yeast strain containing pGal1, 10-IsPETase in the presence of galactose during the time course experiment. The experimental control, BY4741 yeast strain containing pGal1, 10-eGFP, either in glucose or galactose, should not show any TPA product increase due to lack of the IsPETase. However, the team did detect a small amount of TPA products in both co-cultured mediums. A similar unclear pattern of TPA products was observed in BY4741 containing pGal1,10-IsPETase either in glucose or galactose.

Biosafety

For creating the wild-type IsPETase along with several mutations cloned downstream of the T7 promoter to create composite parts and transforming the 4 different composite parts into bacteria, the DE3BL21 strain for the protein expression. However, DH5ɑ bacteria was only used for the entire cloning process to generate more plasmids the team constructed. The wild-type IsPETase cloned downstream of the pGal1,10 promoter to create one composite part and transformed it to the yeast strain, Saccharomyces, which was used in the project, and all of them were microorganisms that only required a P1 level lab. The backbone of the T7 promoter contained the origin of the replication for bacteria to make more copies during mitosis. The backbone of the pGal1,10 promoter contained not only the bacterial origin but also a 2um of the origin of the replication for yeast to make more copies during mitosis. Other organisms receiving our team’s composite parts would not be able to replicate these genes because they had different origins of replication, which meant that our genes would be unable to replicate outside of our controlled experimental conditions. After completing cloning and functional experiments, our team ensured safety by either bleaching the bacteria and yeast plates over the weekend or autoclaving the plates to completely eradicate any bacteria and yeast containing the composite parts.

Citations

Anamika Kushwaha, Goswami, L., Mamata Singhvi, & Beom Soo Kim. (2023). Biodegradation of poly(ethylene terephthalate): Mechanistic insights, advances, and future innovative strategies. Chemical Engineering Journal, 457, 141230–141230. https://doi.org/10.1016/j.cej.2022.141230

Bertocchini, F., & Arias, C. F. (2023). Why have we not yet solved the challenge of plastic degradation by biological means?. PLoS biology, 21(3), e3001979. https://doi.org/10.1371/journal.pbio.3001979

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. https://doi.org/10.1002/elsc.202100105

Maity, W., Maity, S., Bera, S. (2021). Emerging Roles of PETase and MHETase in the Biodegradation of Plastic Wastes. https://doi.org/10.1007/s12010-021-03562-4

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. https://doi.org/10.3390/polym15071779

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. https://doi.org/10.1021/acscatal.9b00568

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O'Connor, C. (n.d.). 13.1: Regulation of the GAL1 promoter. (2014). Libretexts. https://bio.libretexts.org/Bookshelves/Cell_and_Molecular_Biology/Book%3A _Investigations_in_Molecular_Cell_Biology_(O'Connor)/13%3A_Protein_overexp ression/13.01%3A_Regulation_of_the_GAL1_promoter