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Experiment Aims
Plastic, being the cheapest and thus most commonly used packaging materials, poses a grave environmental threat when its disposal is not properly managed. Our team’s aim is to create a genetically engineered mutant IsPETase that is capable of degrading PET plastic more efficiently than current industry methods, as well as more compatible to function at a wider range of conditions. We believe this would encourage adoption of bio recycling and reduce the amount of plastics and microplastics that are in the ocean.
Choosing Target Genes
PETase
PETase is an enzyme that belongs to the esterase class of enzymes. It catalyzes the breakdown of polyethylene terephthalate (PET) plastic through hydrolysis, resulting in the formation of monomeric mono-2-hydroxyethyl terephthalate (MHET), BHET (bis(2-hydroxyethyl) terephthalate), and terephthalic acid (TPA), opening up possibilities for biological PET recycling and degradation. The most popular PETase enzyme was identified in 2016 from a strain of bacteria called Ideonella sakaiensis 201-F6, found in sludge samples collected near a PET bottle recycling site in Japan (Yoshida,2021). IsPETase studied by Japanese groups for several years showed that it has more tolerance for catalytic activities to degrade PET plastic in high temperatures, and acidic environments (Yoshida,2021). 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 (Khairul et al, 2022). One of the key factors to this is the enzyme's structure, in which IsPETase has a broader and more open active-site cleft compared to other enzymes like cutinases, which allows it to accommodate PET molecules more effectively. The wider substrate-binding pocket of the PETase is critical for PET hydrolysis, which is the major process of breaking down plastics into monomeric blocks. Furthermore, IsPETase possesses two disulfide bonds in its active site, contributing to its efficiency in PET substrate binding. After many studies and experiments, IsPETase is 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 and Antibiotic Resistant Gene
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 a high efficiency of RNA transcription of the gene cloned downstream of the T7 promoter, when the promoter is induced by isopropyl β-D-Thiogalactoside (IPTG). It 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 (T7 Promoter System, 2016). The ampicillin-resistant gene is also synthetic and cloned into the T7 promoter plasmid for bacterial transformation selection on LB-Amp plates.
pGal 1, 10 Plasmid and Antibiotic Resistant Genes
In yeast, the promoters of Gal1 and Gal 10 are named after the genes they regulate, GAL1 and GAL10, which encode enzymes involved in the conversion of galactose into glucose-1-phosphate, a form that can be further metabolized by Saccharomyces cerevisiae, yeast cells(O’Connor, 2014). Scientists engineered yeast Gal1 and Gal 10 promoters to create a Gal 1,10 promoter on the plasmid. This 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. Our team will adopt this pGal promoter mainly due to its high expression of the genes (20-30 fold induction) related to this project cloned downstream of the pGal promoter in the presence of galactose for the in vivo protein expression in yeast (Rajeshkannan et al, 2022).
The pGal1,10-SPT5-Streptavidin binding protein (SBP) plasmid, containing yeast origin of replication, 2u, and also bacteria origin of replication, was sponsored by Dr. Tien-Hsien Chang at the Genomics Research Center of Academia Sinica, in Taipei. The reason our team is using this pGal1,10 promoter plasmid is due to the benefit of making biobricks in bacteria and performing the in vivo protein functional assay in saccharomyces yeast. There are two resistant genes on this plasmid, one is the Geneticin (G418) resistance gene which can be used to select yeast colonies on YPD-G418 plates since G418 blocks the protein translation process. Only yeast containing our pGal1,10 plasmid can survive on YPD-G418 plates ; the other is ampicillin, which shows no effect on yeast, but can be used to do bacterial colony selection on LB-Amp plates.
IsPETaseS121E/D186H/R280A Mutations
Japanese scientists did further investigations to show that after changing 3 amino acids at the catalytic site on the PETase enzyme, (IsPETase S121E/D186H/R280A), the mutant enzyme enhances the PETase activity compared to the wild-type PETase, and also can have catalytic activity at higher temperatures to degrade PET plastic products faster (Brett et al, 2021; Maity et al, 2021; Son et al, 2019). The 3 amino acid mutations our team aimed to create will widen the substrate binding cleft, allowing the catalytic activities to perform easier and faster, enhancing the overall efficiency of PET product recycling (Office U.S.G.A, 2022). Therefore, our team’s will focus on enhancing the PETase enzyme via site-directed mutagenesis technique to create (IsPETaseS121E/D186H/R280A) for PET plastic degradation
Design Goal
Our team’s final goal is to focus on enhancing the PETase enzyme activity via site-directed mutagenesis technique to create (IsPETaseS121E/D186H/R280A) for more efficient PET plastic degradation. We will create two versions, one with a pGal1,10 promoter to increase enzyme production, for yeast, and one with a T7 promoter with a his-tag to allow for in vitro protein purification, in E. coli.
Fig 1: pGal 1,10-IsPETase target plasmids
Fig 2: T7-IsPETase target plasmids
Creating our Genetically Engineered Composition Parts
Making T7-IsPETase plasmid
Fig. 3: Making T7-IsPETase plasmid diagram
PCR via PET synthesized from a biotech company
We synthesized the IsPET gene via PCR protocol. We designed the forward primer flanked with the BamHI enzyme recognition site and the reverse primer flanked with the HindIII enzyme recognition site. Our team purchased the synthesized IsPETase gene from the Mission Biotech Company as the DNA template, and a PCR experiment was performed to amplify the IsPETase gene flanked with BamHI and HindIII enzyme digestion sites at both ends of the gene.
Double enzyme digestions on PCR products
We performed double enzyme digestion using the BamHI and HindIII enzymes simultaneously on the IsPETase gene PCR product and the T7 promoter plasmid, which generates compatible sticky ends on both the IsPETase gene PCR product and the plasmid, allowing them to be ligated together.
T4 ligation
We used T4 DNA ligase to ligate the digested IsPETase gene PCR product and the digested T7 Promoter plasmid together. The T4 ligase catalyzes the formation of phosphodiester bonds between the DNA fragments, joining them together to form the T7-IsPETase plasmid.
Bacterial transformation
We transformed the ligated T7-IsPETase plasmid into DH5ɑ bacterial competent cells, and did LB-amp plate selection. The bacterial colonies grew on the LB-amp plates indicating that they contain our team’s cloned plasmids. This was done by heat-shocking the bacterial competent cells with the cloned plasmids and then allowing them to recover and grow on LB-amp selected plates.
We then inoculated those bacterial colonies on LB-amp plates and broth. This allowed the bacteria to grow and multiply, amplifying the cloned DNA in our team’s T7-IsPETase plasmid. We then plated the transformed bacteria on LB-AMP plates and broth. The ampicillin in the plates selected bacteria that have taken up the plasmid, as the plasmid contained an ampicillin resistance gene. Our team picked those single bacterial colonies and stored them in 25% glycerol stocks at -80 °C.
BACTERIAL PLASMID EXTRACTION
After bacterial inoculation, we did bacterial plasmid extraction: our team used a TENs buffer, which contained a more basic solution (0.1M NaOH/0.5% SDS in TE, PH=8), and 10% SDS to break down bacterial cell membranes. Then, we added 3M sodium acetate (NaOAc) to neutralize the solution to precipitate down debris. Then, we used RNase to break down the remaining RNA in the tube and incubated them at 37°C for at least 20 minutes. Next, we used phenol-chloroform and separated the proteins and debris from our plasmids. Finally, we added ethanol and sodium acetate to precipitate the plasmid at the bottom of the tube and poured out the supernatant to obtain our pure, desired plasmid.
PCR Verification
We did PCR again to confirm bacterial colonies containing our team’s T7-IsPETase plasmid: After selecting colonies on the LB-AMP plates, we performed PCR using primers specific to the cloned IsPETase gene to confirm the presence of the desired insert in the plasmid. This helped verify the success of the cloning process (amplification of the targeted DNA segment).
Proof of Concept
Fig. 4: IsPETase full length product, 893bp.
After the team amplified IsPETase with two enzyme cut sites, BamHI1 and HindIII, flanked on the two sides via primers designed through PCR. After PCR, the team ran a sample through 1% agarose gel, IsPETase PCR product showed a single distinct DNA band with a length of 893 base pairs+20 bp of enzyme cut sites designed on primers and showed a nice single band data after PCR amplification. The DNA fragment corresponded with DNA ladders according to the gel electrophoresis.
Fig. 5: IsPETase PCR product
After cloning IsPETase downstream of the T7 promoter plasmid, bacterial transformation was done, and bacterial colonies were grown on the LB-Amp selection plates. The colonies were directly inoculated to perform the PCR technique via the IsPETase primer set. In Fig2, the 1% agarose gel showed that bacterial colonies # 1, 2, and 5 had a distinct IsPETase single PCR product band, which indicated that the bacterial colonies contained T7-IsPETase
Making the pGal 1,10-IsPETase Plasmid
Fig. 6: Making the pGal1,10-IsPETase plasmid
For making pGal 1,10-IsPETase, we performed similar experiments as that of making T7-IsPETase. However, when performing double enzyme digestion, we used XmaI and SpeI double enzymes, instead of BamHI and HindIII enzymes used during double enzyme digestion for T7-IsPETase. 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.
Proof of Concepts
After our experiments, we did PCR again to confirm the yeast contained our team’s pGal1,10-IsPETase plasmid:
Fig.7: pGal 1,10-IsPETase full-length product, 893bp
In order to amplify the IsPETase with two enzyme cut sites, XmaI and SpeI, flanked on the two sides via primers designed, the team performed PCR. After PCR, the team ran a sample through 1% agarose gel, IsPETase PCR product showed a single distinct DNA band with a length of 893 base pairs+20 bp of enzyme cut sites designed on primers and showed a nice single band data after PCR amplification. The DNA fragment corresponds with DNA ladders according to the gel electrophoresis.
Fig. 8: pGal1,10-IsPETase PCR product
After cloning IsPETase downstream of the pGal1,10 promoter plasmid, bacterial transformation was done, and bacterial colonies were grown on the LB-Amp selection plates. The colonies were directly inoculated to perform the PCR technique via the IsPETase primer set. In Fig4, the 1% agarose gel showed that bacterial colonies #5 and 6 had a distinct IsPETase single PCR product band, which indicated that the bacterial colonies contained pGal 1,10-IsPETase
Mutating the Plasmids - Site-Directed Mutagenesis
Fig. 9: T7-IsPETaseS121E Plasmid Creation Diagram
Amplification of mutant DNA
To mutate our IsPETase, we designed point mutations on the forward and reverse primers, changing IsPETase serine 121 to glutamic acid. Next, we performed PCR amplification: for the PCR, prepare the T7-IsPETase a DNA template, pfu DNA polymerase buffer, pfu DNA polymerase, dNTP mix, and the mutated forward and reverse primers. The mutated plasmid was amplified after PCR. Thus, we obtained copies of the mutated plasmid.
Degradation of parental DNA
After the PCR, we separated the mutated plasmid, T7-IsPETaseS121E plasmid, from the parent plasmid by adding 1μl of DpnI to the PCR reaction at 37 °C for 1 hour. This allowed the DpnI to digest the parental DNA, which contains methylation markers, leaving only the T7-IsPETaseS121E plasmid.
Bacterial Transformation into E. coli
We transformed the mutated plasmid into DH5α bacteria competent cells via 42 °Cheat shock protocol on LB-amp selection plates. Then, we prepared it for LB-AMP plate selection, and the resulting bacteria were the E. coli that contained the T7-IsPETaseS121E plasmid. By performing bacterial transformation, we obtained more single bacterial colonies that contained the T7-IsPETaseS121E plasmid.
Inoculate bacterial colonies
We inoculated several bacterial colonies on LB-amp plates and broth; by doing this, we could select the colonies that have the T7-IsPETaseS121E plasmid with an amp-resistant gene. We plated the transformed bacteria on LB-AMP plates and broth. The ampicillin in the plates selected the bacteria that have taken up the T7-IsPETaseS121E plasmid, as the plasmid contains an ampicillin resistance gene, killing the bacterial colonies that do not contain the plasmid. That confirmed that our bacterial colonies contained the T7-IsPETaseS121E plasmid.
Bacterial plasmid extraction
After we got more copies of bacteria cells containing the mutated plasmid, we used the bacterial plasmid extraction protocol to extract the mutated plasmid from the bacteria cells. Our team used TENs buffer that contained a more basic solution and 10% SDS to break down bacterial cell membranes and then 3M NaOAC pH5.2 was added to neutralize the solution to precipitate down protein and membrane debris. RNase was used to break down all of the RNA in the tube and incubate at 37 °C for 20-30 mins. We then used phenol-chloroform and separated the proteins and debris from our plasmids. After extracting the top layer of the remaining mixture, we added ethanol and 3M sodium acetate to precipitate the plasmid at the bottom of the tube. In this stage, the ethanol interacted with the plasmids and helped the plasmids to precipitate at the bottom of the tube. We then spun down the mixture and discarded the supernatant, leaving only the pellet. Again, we added ethanol to clean up the chemicals attached to the plasmids. Finally, we spun down the mixture and discarded the supernatant, and the pure, desired plasmid pellet will be at the bottom of the tube. We then resuspended it with dH₂O. We repeated site-directed mutagenesis two more times to get triple mutations on the IsPETase.
Checking the sequence
After completing each site-directed mutagenesis, we sent our mutant plasmids to Mission Biotech to check our mutation results.
The results showed that our team failed to create the desired mutations. No mutations were on the pGal1,10-IsPETase (BBa_K5094010), however it confirmed we had achieved the pGal1,10-IsPETase (BBa_K5094010) sequence in our plasmid.
Similarly the desired mutations were also not found on the T7-IsPETase (BBa_K5094006). However we had created new mutations: T7-IsPETaseThr116A(BBa_K5094007), T7-IsPETaseThr116A/k259Glu (BBa_K5094009), and T7-IsPETaseThr116A/M154Thr (BBa_K5094008) instead.
Nevertheless, we decided to continue the project, as other publications showed alternative mutations in the IsPETase gene which also enhanced IsPETase enzyme activity(Sevilla et al, 2023). Therefore, our new mutations may also affect the catalytic activity of the IsPETase. The pGal1,10 promoter plasmid we engineered with IsPETase should have a 20-30 fold increase in gene expression of the gene cloned downstream of the promoter, when induced by galactose, which is a valuable design (O’Connor).
Based on the promise of these factors, we decided to continue to determine proof of concept and designed functional assay experiments that would test the functionality of our new mutations in our bacteria and the wild type pGal1,10-IsPETase.
Proof of Concept
Fig. 10 Before sending out sequencing to show the mutations of the IsPETase team expected, our team ran the plasmids on 1% agarose gel. Lane 2 is pGal1,10-IsPETase, lane 3 is pGal1,10-IsPETase S121E, lane 4 is pGal1,10-IsPETase S121E/D186H and lane 5 is pGal1,10-IsPETase S121E/D186H/R280A.
Checking Our Products/Functional Assays
RT-qPCR
Fig. 11: RT-qPCR Time Course Sample Collection Experiment Design
After site-directed mutagenesis, the team couldn’t make any mutations in IsPETase. However, the team decided to use the pGal1,10-eGFP composite part created by the 2022 KCIS team as a control and test if our wild-type of pGal1,10-IsPETase produces our desired enzyme. To manipulate our team’s wild-type of the IsPETase enzyme along with the control eGFP cloned downstream of the pGal1, 10 promoter, we transformed the BBa_K418008, which contained pGal1,10-eGFP created by the 2022 KCIS team, and the BBa_K5094010, which contained BBa_K5094001K+BBa_K5094002 (pGal1,10- IsPETase), into wild-type Saccharomyces Yeast Strain, BY4741.
The function of each cloned gene to generate these 2 composite parts is as follows:
- BBa_K418008 created by the 2022 KCIS team was transformed into BY4741 to make the BY4741 which contains BBa_K418008, and was used as a control yeast strain in our project. The purpose of using this composite part was to determine the specification. The team wants to verify the gene downstream of the pGal1,10 promoter can be induced in the presence of galactose, but eGFP protein can't degrade PET plastic for the co-culture experiments later on.
- BBa_K5094010 contained BBa_K5094001K+BBa_K5094002 (pGal1,10-IsPETase) was transformed into BY4741 to make the BY4741 containing BBa_K5094010 as the experimental yeast strain in our project. BBa_K5094010 contains a wild-type of the IsPETase encodes for the PETase enzyme specifically degrading PET plastic. The team wants to verify the gene downstream of the pGal1,10 promoter can be induced in the presence of galactose, IsPETase protein.
To further verify whether our team’s composite parts produce mRNA, RT-qPCR technique was operated to detect the mRNA induction of eGFP in BY4741 containing BBa_K418008 as a control, the mRNA induction of IsPETase in BY4741 containing BBa_K5094010 as an experimental sample, via time course sample collection, 0min, 30min, 60min, 90min, 120min in the presence of the 2%YP-galactose medium.
The BY4741 containing the two different composite parts was grown in 150ml of 2%YP-glucose-200 μg/ml G418 until OD600 ~0.4 of the 40 ml of yeast culture medium and taken out as 0 min as a control. Without galactose, the pGal1,10 promoter would not express the downstream genes’ mRNA.
The rest of the culture samples were collected at 5000 rpm for 5 mins and we washed the culture samples with dH₂O twice to eliminate glucose. We then transferred the 2 composite parts of the yeast culture samples to 200 ml of 2%YP-galactose-200 μg/ml G418, for which our team took out 40 ml of yeast culture medium samples at different time courses in the presence of galactose.
SDS Gel Electrophoresis and Western Blot
Fig. 12: IPTG Induction Experiment
Before setting up the SDS page and western blot experiments, our team cultured 50 ml of DE3BL21 bacteria cells which contained T7-IsPETase and our mutated plasmids in the LB broth medium until OD~0.2. Then cell culture was split into one half (25mls) that contained 0.5mM of IPTG at 37 °C for 6 hours and the other half (25mls) that did not have IPTG at 37°C for 6 hours as controls (Mühlmann et al, 2017; Namdev et al, 2019).
To verify whether our composite parts—BBa_K 5094006 containing BBa_K5094000+ BBa_K5094002 (T7-IsPETase), BBa_K5094007 containing BBa_K5094000+BBa_K 5094003(T7-IsPETase Thr116A), BBa_K5094009 containing BBa_K5094000+BBa_ K5094005 (T7-IsPETaseThr116Ala/Lys259Glu), and BBa_K5094008 containing BBa_K5094000+BBa_K5094004 (T7-IsPETaseThr116Ala/Met154Thr )—can express the proteins our team desires in DE3BL21 bacteria, the team did whole cell protein extraction of the DE3BL21 bacteria after being induced by IPTG, 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 were collected for a SDS page and Western blot (Team:Harvard, 2017).
The team performed the SDS page first. We ran an 18% SDS-PAGE gel to confirm whether the whole cell extract contained all proteins, as well as to look for a stronger band of the IsPETase along with various mutants of IsPETase protein, approximately 30 kDa in the presence of IPTG.
After 18% SDS-PAGE gel data, our team performed the western blot technique to determine the specific IsPETase protein expression. The team cloned the IsPETase gene downstream of the 6-his tag. Once the IsPETase protein is expressed, it contains a his-tag at the N-terminal of the IsPETase, so the his-tag antibody could be used for the western blot experiment.
Co-Culture Experiment
Fig.13: Co-Culture Experiments Design
The more direct functional assay is to co-culture the same amount of PET film with either DE3BL21 bacteria containing T7-IsPETase plasmid and our mutations on IsPETase enzyme, respectively in the presence of ITPG to induce T7 promoter in DE3BL21 as experimental samples, lack of ITPG as controls. The team used a Nabi machine with 240 wavelengths to detect the amount of TPA, which is the product of PET plastic after degradation, generated daily for the co-culture experiment(Terephthalic acid, 2023). The same experiment was performed with yeast containing the pGal1,10-IsPETase plasmid, along with various mutations on IsPETase, in the presence of galactose as experimental samples, in the glucose as controls.
Future experiments to extend this project:
For the in vitro functional assay:
In addition to in vivo experiments, our team will extend this project to perform in vitro protein purification to isolate the IsPETase enzyme and its various IsPETase enzyme mutants. The IsPETase gene and its mutants are cloned downstream of a T7 promoter engineered with a 6-histidine (6-his) tag. These constructs are then transformed into BL21(DE3) bacteria, and enzyme expression is induced using IPTG.
Following cell lysis, the protein extracts containing the 6-his-tagged wild-type and mutant IsPETase enzymes are passed through a Ni²⁺-resin column, which selectively binds to the 6-his tag. The column is then washed with several buffers to remove unbound and unwanted proteins. Finally, an elution buffer is used to purify the desired enzymes, which are collected in separate tubes for further analysis. The team will use IsPETase enzyme and mutant IsPETase enzymes to perform the enzyme-abstract “p-Nitrophenyl Esters Assay”, which PETase can be tested using p-nitrophenyl butyrate, which releases p-nitrophenol upon hydrolysis by PETase, which can be quantified spectrophotometrically by measuring the absorbance at 405 nm.
Making MHETase biobricks:
The MHET produced from PET degradation by PETase can be further broken down into terephthalic acid (TPA) and ethylene glycol (EG) by MHETase. Our team's future work will focus on designing MHETase biobricks to overexpress this enzyme, aiming to enhance the efficiency of degrading PET plastic into its monomers. This approach will allow for a more complete and efficient breakdown of PET waste, ultimately converting it into reusable building blocks for new plastic production or other industrial applications.
Protocols
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. https://doi.org/10.1002/elsc.202100105
Khairul Anuar, N. F. S., Huyop, F., Ur-Rehman, G., Abdullah, F., Normi, Y. M., Sabullah, M. K., & Abdul Wahab, R. (2022). An Overview into Polyethylene Terephthalate (PET) Hydrolases and Efforts in Tailoring Enzymes for Improved Plastic Degradation. International journal of molecular sciences, 23(20), 12644. https://doi.org/10.3390/ijms232012644
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
Mühlmann, M., Forsten, E., Noack, S., & Büchs, J. (2017). Optimizing recombinant protein expression via automated induction profiling in microtiter plates at different temperatures. Microbial cell factories, 16(1), 220. https://doi.org/10.1186/s12934-017-0832-4
Namdev, P., Dar, H. Y., Srivastava, R. K., Mondal, R., & Anupam, R. (2019). Induction of T7 Promoter at Higher Temperatures May Be Counterproductive. Indian journal of clinical biochemistry : IJCB, 34(3), 357–360.
O’Connor CM. 13.1: Regulation of the GAL1 promoter. (2014). California: Libretexts. https://bio.libretexts.org/Bookshelves/Cell_and_Molecular_Biology/Book%3A_ Investigations_in_Molecular_Cell_Biology_(O%27Connor)/13%3A_Protein_overex pression/13.01%3A_Regulation_of_the_GAL1_promoter.
Office, U. S. G. A. (2022). Science & Tech Spotlight: Biorecycling of Plastics. www.gao.gov. https://www.gao.gov/products/gao-23-106261
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
Tabor S. (2001). Expression using the T7 RNA polymerase/promoter system. Current protocols in molecular biology, Chapter 16, Unit16.2. https://doi.org/10.1002/0471142727.mb1602s11
Team:Harvard - 2017.igem.org. (2017). Igem.org. https://2017.igem.org/Team:Harvard
Terephthalic acid. (2023). Nist.gov; National Institute of Standards and Technology. https://webbook.nist.gov/cgi/cbook.cgi?ID=C100210&Mask=400
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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
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. https://doi.org/10.1016/bs.mie.2020.12.007
