Mar 18, 2026
Degenerate PCR and sequencing workflow for discovering PET hydrolases from Japanese composts
- Ryo Iizuka1
- 1Department of Biological Sciences, Graduate School of Science, The University of Tokyo
Protocol Citation: Ryo Iizuka 2026. Degenerate PCR and sequencing workflow for discovering PET hydrolases from Japanese composts. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9n9opl3e/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We used this protocol.
Created: March 07, 2026
Last Modified: March 18, 2026
Protocol Integer ID: 276270
Keywords: hydrolase genes from compost dna sample, pet hydrolases from japanese compost, compost dna sample, discovering pet hydrolase, sequencing putative polyethylene terephthalate, dna extraction, degenerate pcr, degenerate pcr amplification, hydrolase gene, pcr amplification, colony pcr, japanese compost, microbiology resource announcement, publication in microbiology resource announcement, putative polyethylene terephthalate, sequencing workflow
Funders Acknowledgements:
Institute for Fermentation, Osaka (IFO)
Grant ID: G-2021-3-047
Japan Society for the Promotion of Science (JSPS)
Grant ID: 22K05310
Japan Society for the Promotion of Science (JSPS)
Grant ID: 25K08915
Mitsui Chemicals, inc.
Japan Science and Technology Agency (JST)
Grant ID: JPMJCR2231
Abstract
This protocol describes a degenerate PCR-based workflow for amplifying and sequencing putative polyethylene terephthalate (PET) hydrolase genes from compost DNA samples. It covers DNA extraction, degenerate PCR amplification, gel extraction, TA cloning, colony PCR, and Sanger sequencing with quality filtering. This protocol accompanies a manuscript accepted for publication in Microbiology Resource Announcements.
Troubleshooting
Sample-specific procedures (Compost 1)
Compost 1 was collected from the Miyakojima City Resources Recycling Center in Okinawa (24°45'15.1" N 125°19'52.2" E) on June 13, 2016. The sample was collected into a sterile 50-mL centrifuge tube, transported on dry ice, and stored at −80°C until DNA extraction within one week of collection.
DNA was extracted using the ISOIL kit (Nippon Gene, Tokyo, Japan) according to the manufacturer's protocol.
PCR was performed using TaKaRa Ex Premier DNA Polymerase (Takara Bio, Kusatsu, Japan) with degenerate primers (Fw: 5′-ATGGMSAACCCSTACGAGCGCGG-3′, Rev: 5′-GWRSGGGCAGKTGSMSCGGTACT-3′) [1]. PCR was performed under the following conditions: initial denaturation at 94°C for 1 min; 30 cycles of 98°C for 10 s, 57°C for 15 s, and 68°C for 25 s.
Sample-specific procedures (Compost 2)
Compost 2 was sampled from Kurkku Fields in Chiba (35°19'33.5" N 139°58'53.4" E) on March 14, 2024. The sample was opportunistically collected into a clean paper cup, transported at ambient temperature, and stored at 4°C until DNA extraction.
DNA was extracted by bitBiome Inc. (Tokyo, Japan) using the QIAamp PowerFecal Pro DNA Kit (QIAGEN, Hilden, Germany) according to the manufacturer's protocol.
PCR was performed using TaKaRa Ex Premier DNA Polymerase with degenerate primers (Fw: 5′-ATGGMSAACCCSTACGAGCGCGG-3′, Rev: 5′-GWRSGGGCAGKTGSMSCGGTACT-3′) [1]. PCR was performed under the following conditions: initial denaturation at 94°C for 1 min; 30 cycles of 98°C for 10 s and 68°C for 30 s.
General procedures
The resulting ~800–900 bp amplicons were excised from agarose gels and purified using the NucleoSpin Gel and PCR Clean-up (MACHEREY-NAGEL, Düren, Germany) according to the manufacturer's protocol.
The purified amplicons were re-amplified using TaKaRa Ex Taq Hot Start Version (Takara Bio, Kusatsu, Japan) with the same primer set under the following conditions: 30 cycles of 98°C for 10 s, 55°C for 30 s, and 72°C for 1 min.
The amplified products were purified using NucleoSpin Gel and PCR Clean-up and cloned into the pTAC-1 vector using the FEWBlue TA Cloning Kit (BioDynamics Laboratory, Tokyo, Japan) according to the manufacturer's protocol. Escherichia coli XL10-Gold competent cells were then transformed with the ligation products.
Inserts were confirmed by colony PCR using the KOD One PCR Master Mix (Toyobo, Osaka, Japan) with vector-specific primers (Fw: 5′-GTAAAACGACGGCCAGT-3′, Rev: 5′-CAGGAAACAGCTATGAC-3′) under the following conditions: 30 cycles of 98°C for 10 s, 55°C for 5 s, and 68°C for 1 s. Only clones with the expected insert size (~1,000 bp) were selected for sequencing to ensure the recovery of target PET hydrolase genes.
The colony PCR amplicons were purified using NucleoSpin Gel and PCR Clean-up and sequenced bidirectionally using the vector-specific primers described in Step 10 by the Sanger method at Eurofins Genomics K.K. (Tokyo, Japan) using a 3730xl DNA Analyzer (Thermo Fisher Scientific, Waltham, MA) with the BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific).
Sequencing quality was assessed by visual inspection of chromatograms using ApE software (v2.0.53 and v3.1.4; https://jorgensen.biology.utah.edu/wayned/ape/) [2]. Forward and reverse reads were assembled into consensus sequences, and clones that yielded ambiguous assemblies or contained frameshifts that disrupted the open reading frame were excluded from further analysis.
Homologous protein searches were performed for each deduced amino acid sequence using BLASTP against the NCBI ClusteredNR database (https://blast.ncbi.nlm.nih.gov/Blast.cgi).
For phylogenetic analysis, multiple sequence alignment was performed using ClustalW (with default parameters) in MEGA software (v12.1.2) [3, 4], and a phylogenetic tree was constructed using the neighbor-joining method with 1,000 bootstrap replicates. For visualization, an independent alignment of the deduced amino acid sequences of the 53 amplicons with four reference PET hydrolases (LCC, TfCut2, PHL7, and PHL1) was performed using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) [5] and displayed using ESPript 3.0 (https://espript.ibcp.fr/ESPript/ESPript/) [6].
Protocol references
[1] Sonnendecker, C., Oeser, J., Richter, P. K., Hille, P., Zhao, Z., Fischer, C., Lippold, H., Blázquez-Sánchez, P., Engelberger, F., Ramírez-Sarmiento, C. A., Oeser, T., Lihanova, Y., Frank, R., Jahnke, H-G., Billig, S., Abel, B., Sträter, N., Matysik, J., and Zimmermann, W. Low Carbon Footprint Recycling of Post-Consumer PET Plastic with a Metagenomic Polyester Hydrolase. ChemSusChem 15, e202101062 (2022).
[2] Davis, M. W., and Jorgensen, E. M. ApE, A Plasmid Editor: A Freely Available DNA Manipulation and Visualization Program. Front. Bioinform. 2, 818619 (2022).
[3] Kumar, S., Stecher, G., Suleski, M., Sanderford, M., Sharma, S., and Tamura, K. MEGA12: Molecular evolutionary genetic analysis version 12 for adaptive and green computing. Mol. Biol. Evol. 41,1–9 (2024).
[4] Stecher, G., Suleski, M., Tao, Q., Tamura, K., and Kumar, S. MEGA 12.1: Cross-platform release for macOS and Linux operating systems. J. Mol. Evol. 94,14–18 (2026)
[4] Sievers, F., Wilm, A., Dineen, D., Gibson, T. J., Karplus, K., Li, W., Lopez, R., McWilliam, H., Remmert, M., Söding, J., Thompson, J. D., and Higgins, D. G. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 7, 539 (2011).
[5] Robert, X., and Gouet, P. Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res. 42, W320–W324 (2014).
Acknowledgements
This study was supported by JSPS KAKENHI Grant Numbers 22K05310 and 25K08915, the Institute for Fermentation, Osaka (no. G-2021-3-047), JST CREST Grant Number JPMJCR2231, and the collaboration between The University of Tokyo and Mitsui Chemicals, Inc.