Rifqi Zahroh Janatunaim and Azzania Fibriani* Pages 1 - 6 ( 6 )
Aims: To produce recombinant E.coli that carry plastic degradation enzyme (MHETase)
Background: Plastics have a flexibility that can be formed into solid objects to produce various shapes and sizes (Vert et al., 2012). It can be used at various temperatures because the chemical properties are resistant to light, very strong and tough and easily formed at high temperatures with low production prices (Andrady and Neal, 2009). Polyethylene terephthalate (PET) is the most widely produced polyester plastic in the world. There were 7.4% of plastics converted into PET polymers from total world plastic production in 2016 (PlasticsEurope, 2017). PET is very difficult to catalyze or biological depolymerization due to limited access to ester bonds. Whereas, chemical recycling has been carried out because of the high processing costs compared to PET production. Consequently, plastic will be stockpiled or flowed into the environment which is projected until hundreds of years (Gregory and Anthony, 2003). In 2016, Shosuke Yoshida and team, were discovered that Ideonella sakaiensis 201-F6 has the ability to degrade PET and able to use PET as their main source of energy and carbon. This discovery give the opportunities for researchers to increase the potential bacteria in producing the PET degrading enzyme. In previous study PETase gene has been constructed in pUCIDT plasmid with native signal peptide from Ideonella sakaensis 201-F6 and constitutive promoter J23106 was expressed in Escherichia coli BL21 (DE3). Expression analysis using SDS-PAGE demonstrated that PETase was expressed in extracellular and intracellular. By Response surface methodology (RSM)-based modeling was known that temperature and pH optimum of PETase activity were 38.8°C and 7.7 respectively. However, PET final product, ethylene glycol and terephthalic acid, can be achieved by MHETase degradation to the intermediet product. Hence, in this study MHETase will be constructed in the same construction and host system as PETase recombinant enzyme.
Objective: To construct and to clone recombinant enzyme MHETase that can be used in plastic degradation process.
Materials and Methods: MHETase synthetic plasmid, Escherichia coli BL21 (DE3) bacterial and competent cell, Luria-Bertani agar and broth media, purification plasmid kit GeneAid®, absolut ethanol, 70% ethanol, spiritus, ice cube, 105 ppm ampicilin, cuvet, 1% SDS and deion. Construction of MHETase Gene MHETase sequence were obtained from UniProtKB amino acid database (https://www.uniprot.org) with access number A0A0K8P8E7. Then, the sequence was translated into nucleotide sequences using EMBOSS (https://www.ebi.ac.uk › Tools › emboss_transeq). The sequences was aligned using BLASTp (https://blast.ncbi.nlm.nih.gov) for sequence confirmation. The sequence was used to construct the MHETase expression system in the ampicillin-resistant pUCIDT plasmid (pUCIDT-Amp+). The model of gene and plasmid was constructed in silico using SnapGene software. Synthesis of MHETase carried out with Integrated DNA Technologies (IDT) synthesis services. Plasmid Transformation to Escherichia coli BL21 (DE3) The transformation pUCIDT plasmid contain MHETase gene to E. coli BL21 (DE3) competent cells were done by heat shock method. An amount of 2 μL (20 ng/μL) plasmid were added in 50 μL competent cells. The cells were incubated in ice for 30 minutes. After incubation, the heat shock was carried out by moving the cell into 42° C waterbath for 90 seconds followed by transfer back to the ice directly and incubated in ice for 5 minutes. Then, the 800 μL of Luria-Bertani medium cells were added and cells were grown in incubator shaker at 37 ° C for 1.5 hours. The culture was centrifuged at 14,000 rpm for 2 minutes. A total 750 μL supernatant was discarded and 50 μL used for platting on Luria-Bertani agar medium with 105 ppm ampicillin. The isolate was incubated at 37 ° C for 16 hours. Plasmid Isolation and Sequencing MHETase gene were amplified from E. coli BL21 (DE3) containing transformed plasmid using synthetic primer (forward primer 5’- GGTATAGTGCTAGCAAAGA-3’, reverse primer 5’- CGCTACTAGTATATAAACGC-3’). PCR was performed in 52 ºC annealing and 10 µL PCR mix. Then, amplified product was separated by 1% agarose gel electrophoresis. The DNA product of the correct size (1943 base pairs) was purified using GeneAid Presto™ Purification Plasmid Kit and subjected for sequencing by Macrogen. Sequence Alignment The sequence obtained was aligned by SnapGene and MEGA X software, and BLASTp analysis against MHETase plasmid construction.
Result: In the construction phase, MHETase gene protein sequence was obtained from protein database Uniprot with access number A0A0K8P8E7. The conversion of amino acid to nucleotide sequence was synthesized by Escherichia coli codon usage using EMBOSS Trans-Seq and confirmed by BLASTp. The sequence was used as insert in the pUCIDT plasmid with native signal peptide from Ideonella sakaensis 201-F6 and constitutive promoter J23106 as shown in Figure 1. The promoter sequence used was the constitutive promoter sequence J23106. This constitutive promoter is relatively moderate strength and has good performance (Anderson, 2006). In addition, the use of constitutive promoters was aimed to reduce the variables that can affect the modeling of PETase activity. Strength promoters are being used to avoid too fast protein production which can lead to protein folding errors. Incorrect protein folding will inhibit the activity of the protein produced that can reduce the total of PETase activity (Baneyx and Mujacic, 2004). Furthermore, in this construction was used native signal peptide from Ideonella sakaensis 201-F6 as previous study. The terminator used was the T7Te terminator. The terminator will stops transcription by forming hairpin secondary structure, thus RNA polymerase released (Shetty, 2003). The synthesized plasmid was used to transform Escherichia coli BL21 (DE3) cells by heat shock. Host transformation was verified using plasmid isolation techniques followed by PCR plasmid and visualization in electrophoresis gel. The band under 2000 bp against 1 kb marker size was shown in Figure 2. Based on the gene size in database and construction, the band size is 1813 bp. This result has shown positively that the gene successfully contained in the cells. The sequencing result from Macrogen was analyzed using SnapGene was demonstrated that the gene was in-frame in pUCIDT plasmid (Figure 3). The result means that the portion of a DNA sequence that, when translated into amino acids, contains no stop codons. Therefore it can be translate to mRNA and is being read by ribosomes to make a protein (NIH, 2019). Furthermore, in the analysis pairwise alignment using MEGA X and NCBI was shown that MHETase sequence is totally same as construction gene. Protein BLAST NCBI demonstrated that both sequence has 100% similarity and include in MHET hydrolase with accession number A0A0K8P8E7.1 (same as Uniprot code). Accordingly, E. coli BL21 (DE3) contain MHETase gene are expected to be able to produce enzyme proteins for PET plastic degradation testing.
MHETase gene was successfully constructed in plasmids by in silico method. Synthetic plasmids transformed in E. coli BL21 (DE3) contain MHETase gene sequences which were in frame. Hence, the E. coli BL21 (DE3) cells have the potential to produce MHETase proteins for the plastic degradation testing process.
Other: This study was financially supported by Hibah Riset ITB 2019.
PET, MHETase, PETase, plastic, degradation, recombinant
Institute Teknology Bandung, School of Life Science and Technology, bandung, 40132, Institute Teknology Bandung, School of Life Science and Technology, bandung, 40132