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Our team performed the PCR technique to amplify IsPETase using the IsPETase gene synthesized by the Mission Biotech company as the DNA template and then confirmed the step's success by observing the right size bands of the IsPETase gene PCR products on 1% agarose gel in Figure 1. The team designed the forward and reverse primer set flanked with two enzyme cut sites, BamHI1 and HindIII to generate the composite part, BBa_K5094006 (T7-IsPETase).
Fig 1:BBa_K5094002,IsPETase full length product, 873bp
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 873 base pairs+20 bp of enzyme cut sites designed on primers and Figure 3 showed a nice single band data after PCR amplification. The DNA fragment corresponds with DNA ladders according to the gel electrophoresis.
The team performed bacterial transformation on the LB-Amp selection plates after finishing the cloning steps and inoculated several bacterial colonies to conduct the PCR technique to prove that our team had successfully created a composite part, BBa_K 5094006 (T7-IsPETase) in Figure 2.
Fig2: 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)
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 Figure 2, 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 our team’s composite part, BBa_K5094006 (T7-IsPETase).
Our team not only cloned IsPETase downstream of the T7 promoter plasmid but also pGal1,10 plasmid. The team performed PCR technique to amplify IsPETase using the IsPETase gene synthesized by the Mission Biotech company as the DNA template and then confirmed the step's success by observing the right size bands of the IsPETase gene PCR products on 1% agarose gel in Figure 3. The team designed the forward and reverse primer set flanked with two enzyme cut sites, XmaI and SpeI to generate the composite part, BBa_K5094010 (pGal1,10-IsPETase).
Fig3: BBa_K5094002 IsPETase full-length product, 873bp
After the team amplified IsPETase with two enzyme cut sites, XmaI and SpeI, 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 873 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.
The team performed bacterial transformation on the LB-Amp selection plates after finishing the cloning steps and inoculated several bacterial colonies to conduct the PCR technique to prove that our team had successfully created a composite part, BBa_K5094010 (pGal1,10-IsPETase) in Figure 4.
Fig4: After bacterial transformation for pGal-IsPETase 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)
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 Figure 4, 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 our team’s composite part, BBa_K5094010 (pGal1,10-IsPETase).
Our team's original idea was to make single, double, and triple mutants of the IsPETase downstream of the T7 promoter, and the pGal1,10 promoter respectively. Those mutations were (IsPETase S121E, IsPETase S121E/D186H, and IsPETase S121E/D186H/R280A), since Japanese scientists showed that changing 3 amino acids at the catalytic site on the IsPETase 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 (Son et al paper, and 2021 PET degradation pathway and ref45 on the 2021 paper). To reach the goal of the team’s idea, site-directed mutagenesis was performed, and the desirable mutated amino acid was designed on forward and reverse primers (detailed in the experimental design cycle). Repeat site-directed mutagenesis two more times gave the team the double and triple mutant of the IsPETase. Before sending out for the sequence, the plasmids were run on 1% of the agarose gel, and after receiving the sequence data, the team created alternative IsPETase mutant constructs: T7-IsPETase as a wild-type, T7-IsPETase Thr116Ala, T7-IsPETase Thr116Ala/M154Thr, and T7-IsPETaseThr116Ala/K259Glu on Figure 5.
Fig5: 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-IsPETaseThr116Ala, lane 4 was T7-IsPETaseThr116Ala/M154Thr,and lane 5 was T7-IsPETaseThr116Ala/K259Glu. The plasmids’ conformations were supercoiled, open-circular, and linear on the gel.
After the team did site-directed mutagenesis to make mutations of the IsPETase, the plasmids were run on 1% agarose gel to show in Figure 5 that the team did have the T7 promoter IsPETase, and after sending out sequencing at the Mission Biotech company, the team created T7-IsPETase Thr116Ala, T7-IsPETase Thr116Ala/M154Thr, and T7-IsPETaseThr116Ala/K259Glu. The plasmids’ conformations were supercoiled, open-circular, and linear on the gel.
The pGal1,10 promoter allows for highly efficient expression of our team’s cloned IsPETase gene by adding galactose to the yeast medium for about 20-30-fold induction (2022). Our team utilized the pGal1, 10 promoter in yeast to determine if the IsPETase gene, along with variable mutations of the IsPETase gene cloned downstream of the pGal1,10 promoter, respectively, exhibit different PET plastic degradation levels in the co-culture functional assay.
To reach the goal of the team’s idea mentioned above, site-directed mutagenesis was performed, and the desirable mutated amino acid was designed on forward and reverse primers (detailed in the experimental design cycle). Repeat site-directed mutagenesis two more times gave the team the double and triple mutations of the IsPETase. Before sending out for the sequence, the plasmids were run on 1% of the agarose gel, and after receiving the sequence data, the team realized that we only have pGal1,10- IsPETase plasmid in Fig6, which showed 1% of the agarose gel contained the plasmids, which were all pGal1,10- IsPETase plasmid without any mutations.
Fig6: After sequencing, the team only created pGal1,10-IsPETase plasmid (lanes2,3,4), and lane1 is a 1kb DNA ladder.
After the team did site-directed mutagenesis to make mutations of the IsPETase, the plasmids were run on 1% agarose gel to show the team did have the T7 promoter IsPETase, and after sending out sequencing at the Mission Biotech company, the team created pGal1,10-IsPETase plasmid on lanes2,3, and 4. The plasmids’ conformations were supercoiled, open-circular, and linear on the gel.
The IsPETase gene sequence suppressed our team with 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/M154Thr(BBa_K5094008), and T7-IsPETaseThr116Ala/K259Glu (BBa_K5094009) for DE3BL21 bacteria. pGal1,10-IsPETse (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 (more detail in part section).
To further verify whether our team’s composite parts were biological function, RT-qPCR technique was operated to detect the mRNA induction of eGFP in BY4741 containing BBa_K418008 created by the 2022 KCIS team as a control, the mRNA induction of IsPETase in BY4741 containing BBa_K5094010 as an experimental sample, via timecourse sample collection, 0min, 30min, 60min,90min, 120min in the presence of the 2%YP-galactose medium. The BY4741 contained two different composite parts, respectively, growing in 150ml of 2%YP-glucose-200ug/ml G418, until OD600 ~0.4, and then 40 ml of yeast culture medium was taken out as 0 min as a control. Without galactose, the pGal1,10 promoter would not be induced to express downstream of genes’ mRNA, and the rest of the culture samples were collected, respectively, at 5000rpm for 5 mins and washing the culture samples with dH2O twice to eliminate glucose. Then transferring the 2 composite parts of the yeast culture samples to 200ml of 2%YP-galactose-200ug/ml G418, our team took out 40ml of yeast culture medium samples at, 30’,60’, 90’, and 120’ in the presence of galactose.
To manipulate our team’s wild-type of the IsPETase enzyme with the control eGFP cloned downstream of the pGal1, 10 promoter, respectively. BBa_K418008 contained pGal1,10-eGFP, BBa_K5094010 contained pGal1,10- IsPETase as the team’s experimental sample, were then transformed into wild-type Saccharomyces Yeast Strain, BY4741, respectively. After collecting different time course samples, the whole cell RNA extracted was operated before the RT-qPCR technique was performed.
Fig 7a: 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.
Fig 7b: 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 detect the IsPETase mRNA induction, our team has designed a pair of IsPETase primers within the gene and a common primer set of ACT1 to detect ACT1 mRNA induction as an internal control. BY4741 yeast containing BBa_K418008 (pGal1,10-eGFP), as an experimental control, did show significant eGFP mRNA induction approximately 4-5 fold at 30’,60’, and 90’ in the presence of the galactose compared to the 0’ sample without galactose. The maximum eGFP mRNA induction was around 8-fold at 120’ in the presence of the galactose compared to the sample at 0’ without galactose. Overall, Figure 7a, indicated the team’s experimental design was working properly to use 2% galactose for the pGal1,10 promoter induction.
In the Figure 7b, BY4741 yeast contained BBa_K5094010 (pGal1,10-IsPETase) showed no significant IsPETase mRNA induction at different time course samples in the presence of the galactose compared to the 0’ sample without galactose in Figure 7b. The data indicated that the IsPETase cloned downstream of the pGal1,10 promoter was not induced within 2 hours in the presence of the galactose. For the future experiment, the team will extend the time course sample collection after 2 hours to detect the IsPETase mRNA induction. Commonly, different genes cloned downstream of the pGal1, 10 promoter have different mRNA induction timecourse patterns in the presence of the galactose, and our team clearly showed the experimental design was working in Figure 7a, To further determine an IsPETse mRNA induction timecourse pattern, the team will collect multiple time-course samples in the presence of the galactose behind 2 hours.
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-IsPETaseThr116Ala/Met154Thr), and BBa_K5094009 containing BBa_K5094000-BBa_K5094005 (T7-IsPETaseThr116Ala/k259Glu ), 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 8.
Fig 8: 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-IsPETaseThr116A (-)IPTG, and lane 5 is T7-IsPETaseThr116A (+0.5mM)IPTG. Lane 6 is T7-IsPETaseThr116A/k259Glu (-)IPTG, and lane 7 is T7-IsPETaseThr116A/k259Glu (+0.5mM)IPTG. Lane 8 is T7-IsPETaseThr116A/M154Thr (-)IPTG, and lane 9 is T7-IsPETaseThr116A/M154Thr (+0.5mM)IPTG. IsPETase protein has about 30 kDa molecular weight based on the Harvard 2016 team’s part information.
After the team did the whole cell protein extract with all of the samples (detailed in Fig 8), we ran the protein extract samples on 18% SDS-PAGE gel to show that the team successfully retained the whole protein extract from each sample. In Figure 8, the team labeled the IsPETase protein approximately 30 kDa molecular weight on 18% SDS-PAGE gel. Figure 8, also showed one strong band around 30 kDa in lane 7, T7-IsPETaseThr116A/k259Glu (+0.5mM)IPTG. However, the team wanted to determine the specific IsPETase protein expression at different samples, so we will perform the Western Blot analysis.
The more direct functional assay 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 “ 240nm” 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. 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.
The more direct functional assay 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 “ 240nm” 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. 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 9a: 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.
Our team, originally, was expecting to see an increased trend of TPA product generated in the DE3 bacteria containing T7-IsPETase along with several IsPETase mutants, respectively, after being induced by the IPTG compared to the control samples lack of IPTG, so the team designed the co-cultured experiment. The team added the same amount/shape of the PET film in each co-culture sample medium. To detect the TPA product, the wavelength 240 nm was used to detect the TPA product(Terephthalic acid, 2023). However, in Figure 9a, for the experimental controls, DE3 bacteria only, along with several IsPETase mutants without IPTG generated TPA product, indicated that the culture mediums might have some materials which can also be detected at 240 nm wavelength. For the experimental samples, the DE3 bacteria containing T7-IsPETase along with several IsPETase mutants, respectively, with and without IPTG also had unclear TPA product increase patterns.
Overall, there was no significant increase/any pattern TPA product in the PET-film co-cultured with the DE3 bacteria containing T7-IsPETase, along with several IsPETase mutants, respectively, at the time course data in the presence of IPTG compared to the ones without IPTG.
Fig 9b: 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.
Our team, originally, was expecting to see an increased trend of TPA product generated in the BY4741 yeast strain containing pGal1,10-IsPETase after being induced by the 2% galactose compared to the control sample in the 2%glucose, so the team designed the co-cultured experiment. The team added the same amount/shape of the PET film in each co-culture sample medium. To detect TPA product, the team used the wavelength 240nm to detect the TPA product(Terephthalic acid, 2023) However, in Figure 9b, the experimental control, BY4741 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.
There was no significant increase of the TPA in the PET-film co-cultured with the yeast containing pGal1,10-IsPETase at the time course data in the presence of galactose compared to the one in the glucose. In the final time course, the team did see the TPA was increased in the presence of the galactose, however, the experiment was only done once, and repeating experiments will need to be done. At the same time, our team will also try to smash PET film into powder for the co-culture experiment.
The team has successfully created various mutant IsPETase enzymes and the wild-type IsPETase enzyme cloned downstream of the T7 promoter, respectively, as well as the wild-type IsPETase cloned downstream of the pGal1,10 promoter, which is an impressive accomplishment for a high school student team given the complexity of these tasks. The team has also performed RT-qPCR, Western Blot, and co-culture functional assays to evaluate enzyme expression and activity. However, these assays will require further optimization to produce clear and reliable data, enabling more precise insights into the enzyme's performance.
Here are the ideas the team will use to optimize these functional assays:
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.
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 (as indicated in Fig. 8). 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.
The team cut PET film into uniform circular pieces, adding 15 pieces to each sample for the co-culture assay. However, the results did not meet our expectations. Following an online meeting with our collaborators, it was suggested that we grind the PET into a powder for the co-culture experiment. We plan to implement this recommendation in our next trial.
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.
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.
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.
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.
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.
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.
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.