Part:BBa_K4901036:Design
Expression vector with IsPETase mutant gene
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 2012
Illegal NheI site found at 2035
Illegal NotI site found at 2196 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
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 DH5α 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 DH5α by heat shock and plated on selective LB medium with ampicillin (100 μg/ml).
Next, the presence of vectors (positive colony) with IsPETase_W159H_S238F-6xHis-tag gene was identified by amplification using the PCR method using PCR Master mix (×2) (Thermo Scientific, K0172). For the reaction, we took 50 ng of DNA, 25 pmol of primers (Direct 5'-AACTTCCCCCGTGCC-3' and Reverse 5'-CTCGAGGGAACAGTTCGC-3') and 25 μl of the PCR mixture, the total volume was adjusted to 50 μl. PCR program: Initial DNA denaturation - 94°C, 5 min; subsequent DNA denaturation - 94°C, 30 sec; annealing - 52°C, elongation - 72°C, 1 min and final elongation at 72°C, 5 min. The number of PCR cycles is 30. PCR products were analyzed in a 1.0% agarose gel with the addition of ethidium bromide and visualized under transmitted UV light using a transluminator. 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 1).
[[File:wiki/teams/4901/wiki/figure-1.png]
Figure 1: 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 2.
[[File:wiki/teams/4901/wiki/figure-2.png]
Figure 2. 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.
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
Hydrolysis of p-nitrophenyl butyrate (pNPB)
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 S238F) 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 (Figure 3).
Figure 3 - Analysis of PETase_W159H_S238F PETase enzyme activity using p-nitrophenyl butyrate.
Source
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