Analysis
Enzymatic Modification
References
Analysis of IsPETase_W159H_S238F with 6xHis-tag gene expression
The gene IsPETase_W159H_S238F with 6xHis-tag, which had two mutations and was 3.5-fold more efficient than wild-type PETase, was selected from the listed genes [2]. This gene sequence was located in the vector pSB1C5C (Registry Part ID: Bba_k2910000).
At the initial stages of work, we needed to develop the plasmid pSB1C5C-IsPETase_W159H_S238F-6xHis-tag and the genes of the promoter (BBa_J23118), strong RBS (BBa_K2680529) and constitutive terminator (Bba_J428091) for further work with them. For this purpose, electrocompetent cells of E. coli strain DH5a were used. For analysis, 1 μl of diluted plasmid was taken and mixed in a pre-chilled electroporation cuvette with 40 μl of cells. Next, the electroporation program was set: voltage 1700 V (field strength 17 kV/cm), resistance 200 Ohm, and capacitance 25 μF. The sample was then pulsed once, the cuvette was quickly removed, and 960 μL of SOC medium (kept at 37°C) was immediately added to resuspend the cells. Transfer the cells to a sterile BD Falcon 14 mL conical bottom polypropylene tube and incubate the tube at 37°C for 1 hour with shaking at 225-250 rpm. Next, 100 μl of the transformation mixture was applied to LB agar plates containing the appropriate antibiotic (Chloramphenicol 25 μg/ml).
The colonies obtained the next day were subcultured into 5 ml of LB medium with antibiotic and incubated overnight at 37°C with shaking at 180 rpm. The next morning, plasmids were isolated using the GeneJET Plasmid Miniprep Kit (Thermo Scientific™, K0503). The concentration of the resulting plasmids and purity were checked on Nanodrope One C. The concentration of plasmids was about 150 ng/μl and the purity was 1.8-1.9 (Ratio 260/280).
The resulting plasmids were treated with BsaI restriction enzymes in the Green buffer. 2000 ng of plasmids and 2 unit each were taken for restriction. enzymes for a total volume of 20 µl. The reaction lasted 1 hour at 37°C followed by inactivation at 65°C for 20 minutes. The processed products were then analyzed on a 1.0% agarose gel. After confirmation of the restriction products, ligation was carried out using T4 DNA ligase (Thermo Scientific™, EL0014) at 22 °C for 1 hour. The ligation products were used for transformation. Ligation products (10 μl) were transformed into strain DH5a by heat shock and plated on selective LB medium with ampicillin (100 μg/ml).
The resulting plasmids were treated with BsaI restriction enzymes in the Green buffer. 2000 ng of plasmids and 2 unit each were taken for restriction. enzymes for a total volume of 20 µl. The reaction lasted 1 hour at 37°C followed by inactivation at 65°C for 20 minutes. The processed products were then analyzed on a 1.0% agarose gel. After confirmation of the restriction products, ligation was carried out using T4 DNA ligase (Thermo Scientific™, EL0014) at 22 °C for 1 hour. The ligation products were used for transformation. Ligation products (10 μl) were transformed into strain DH5a by heat shock and plated on selective LB medium with ampicillin (100 μg/ml).
PCR products were analyzed in a 1.0% agarose gel with the addition of ethidium bromide and visualized under transmitted UV light using a transilluminator. As a result, a DNA fragment of 890 nucleotide pairs in size was amplified, corresponding to the length of the IsPETase_W159H_S238F gene with 6xHis-tag (Figure 5).
Figure 5. Analysis of IsPETase_W159H_S238F gene amplification product with 6xHis-tag. M - GeneRuler 1 kb DNA Ladder marker; 1-2 - PCR amplification product.
After confirmation of the correctness of the obtained plasmid, transformation into expression strain TG1 was performed. The obtained colonies were also tested for the presence of the plasmid using specific primers: Direct 5'-AACTTCCCCCCCGTGCC-3' and Reverse 5'-CTCGAGGGAACAGTTCGC-3'. Positive colonies were used for protein induction. Induction conditions: Overnight bacterial culture was crossed into 50 ml at a ratio of 1:50 and incubated at 37°C until OD600 was 0.6. The culture temperature was then reduced to 30°C and 0.5 mM IPTG was added and left overnight at 30°C on a shaker.
The next morning, protein extraction was performed by sonication and the cell extract was analysed by SDS-PAFE electrophoresis using 15% gel. After confirming the presence of protein in the cell extract, induction was carried out in 1L of LB medium. Protein extraction was performed in lysis buffer (50 mM Tris-HCl (pH 9.0), 50 mM NaCl, 20 mM imidazole, 1 mM EDTA, 5 mM β-mercaptoethanol, 1 mM DTT, 2% of Triton X-100, and 5% of glycerol) supplemented with Complete Protease Inhibitor Cocktail (Roche Diagnostics, Switzerland). The resulting supernatant after centrifugation was used for protein purification on an FPLC ÄKTA start chromatography protein purification system using a 1 ml HisTrap column charged with Ni2+. The purified fractions were subjected to gel electrophoresis. The result of gel electrophoresis is shown in Figure 7.
Figure 6. SDS-PAGE gel electrophoresis of IsPETase_W159H_S238F protein with 6xHis-tag. M, protein marker; 2-14, purified fractions from tubes 1 to 13
From the gel electrophoresis result, we can see that we were able to express and purify IsPETase_W159H_S238F with 6xHis-tag (32.4 kDA). This protein can be further analysed for enzymatic activity and further used for plastic recycling.
Enzymatic Modification of PET: Surface Hydrophilization and Polymer Chain Analysis.
Figure 7. Investigating PET hydrolase-mediated hydrolysis for surface hydrophilization and analyzing interactions with polymer chain ends, enhancing our understanding of enzymatic PET modification.
1. Hydrolysis of PET Film to Ends/Loops of Polymer Chains: Polyethylene terephthalate (PET) is a synthetic polymer used in various applications, including plastic bottles and films. The hydrolysis of PET film involves breaking down the polymer chains through a chemical reaction with water. This process can occur in two steps:
a. Hydrolysis: In this step, water molecules (H2O) interact with the ester linkages in PET. The ester linkages are the bonds that hold the polymer chains together. Hydrolysis breaks these ester bonds, resulting in shorter polymer chains, as well as the release of molecules like ethylene glycol (EG) and terephthalic acid (TPA).
b. Ends/Loops of Polymer Chains: The result of hydrolysis is the creation of polymer chains with open ends and loops. The ends of the chains may have functional groups exposed, making them more reactive for subsequent chemical reactions or recycling processes. These ends and loops can be further processed into monomers or intermediates for PET recycling or used to create new PET-based materials.
2. Surface Hydrophilization: PET is known for its hydrophobic nature, meaning it repels water. Surface hydrophilization is the process of modifying the surface of PET to make it more water-attractive or hydrophilic. This can be achieved in several ways:
a. Chemical Treatment: Chemical treatments involve applying substances or coatings to the PET surface that have hydrophilic properties. For example, plasma treatment or chemical grafting can introduce hydrophilic functional groups to the surface, making it more receptive to water.
b. Physical Methods: Physical methods can also be used to enhance hydrophilicity. These methods include roughening the surface through techniques like sandblasting or creating micro/nanostructures that can trap water molecules.
c. Additives and Coatings: Sometimes, hydrophilic additives or coatings are applied to the PET surface to change its properties. These coatings can include surfactants, coatings with hydrophilic nanoparticles, or other materials that interact favorably with water.
By hydrophilizing the PET surface, it becomes more suitable for applications where contact with water is necessary, such as in medical devices, food packaging, or membranes used for water purification.
Hydrolysis of p-nitrophenyl butyrate (pNPB)
In the experiment described, the hydrolysis of p-nitrophenyl butyrate (pNPB) was studied using two enzymes: IsPETase and wild-type (WT) enzyme. The reaction conditions included a substrate concentration of 2500 uM (micromolar), a slightly basic pH of 9.0, and a temperature of 30°C.
During the experiment, the enzymes catalyzed the hydrolysis of pNPB, breaking it down into p-nitrophenol and butyric acid. We used this reaction to understand the kinetics and substrate specificity of enzymes. The specific purpose of this experiment is investigating the efficiency of IsPETase compared to the wild-type enzyme, studying the enzyme's activity under specific pH and temperature conditions, and exploring potential applications in biotechnology.
In the experiment comparing PETase wildtype with the variant BBa_J435500_W159H_S238F, the researchers assessed their respective enzymatic activities under specific conditions. The experiment focused on the hydrolysis of p-nitrophenyl butyrate (pNPB) at a concentration of 2500 nanomolar, with the reaction taking place at pH 9.0 and a temperature of 30 degrees Celsius. The results demonstrated a remarkable difference in enzymatic activity between the two variants.
Wildtype PETase: The wildtype PETase exhibited a relatively lower enzymatic activity under the specified conditions. The graph representing the wildtype PETase's activity showed a gradual increase in product formation (p-nitrophenol) over time. However, the rate of reaction was comparatively slower.
BBa_J435500_W159H_S238F: In contrast, the variant BBa_J435500_W159H_S238F displayed significantly enhanced enzymatic activity. The graph depicting its activity revealed a much steeper slope, indicating a rapid conversion of pNPB into p-nitrophenol. The variant exhibited a catalytic efficiency that was seven times higher than that of the wildtype PETase under the same conditions.
This significant improvement in enzymatic activity in the BBa_J435500_W159H_S238F variant can be attributed to the specific mutations (W159H and IsPETase) incorporated into its structure. These mutations likely enhance the substrate-binding affinity and catalytic efficiency of the enzyme, leading to the observed increase in activity. Overall, the results highlight the potential for using BBa_J435500_W159H_S238F variant in applications requiring efficient enzymatic hydrolysis of PET (polyethylene terephthalate) or similar substrates, as it outperforms the wildtype PETase by a substantial margin under the specified experimental conditions.
To evaluate the activity of expressed PETase enzyme in comparison to the original wild type and our BBa_J435500_W159H_S238F PETase variant, we conducted assays using p-nitrophenyl butyrate. This compound mimics the ester bonds found in PET plastic. When PETase enzymes break these ester bonds, they release a signal at 405 nm, so we observed absorbance at 405 nm. Additionally, to compare the activity of wild type, we used the result of wild-type PETase activity from iGEM registry parts (Part: BBa_K2910000) designed by iGEM 2019 Toronto team. The graph above demonstrates the outcomes of these assays for the mentioned PETase variants.
References
1. Majid H. Al-Jailawi, Rasha S. Ameen and Ali A. Al-Saraf (2015). Polyethylene degradation by Pseudomonas putida S3A. Int. J. Adv. Res. Biol.Sci. 2(1): (2015): 90-97
2. Writtik Maity, Subhasish Maity, Soumen Bera, Amrita Roy (2022). Emerging Roles of PETase and MHETase in the Biodegradation of Plastic Wastes. Applied Biochemistry and Biotechnology https://doi.org/10.1007/s12010-021-03562-4