Dna polymerase

All chemicals used were reagent grade. Restriction endonucleases, DNA ligase, and DNA polymerase were purchased from NEB (MA, USA). Primers were synthesized by Xcelris (India). All chromogenic substrates and ionic liquids were purchased from Sigma-Aldrich. The active fractions post-purification were pooled and concentrated using 30 kDa cut-off size membranes of Amicon-Ultra-15 (Millipore).

The strains, plasmids, and primers were defined in Supplementary Tables 1–3, respectively. The plasmids were constructed according to the conventional molecular biology techniques, such as PCR, DNA restriction, ligation, transformation, and positive colony selection (Aviv and Gal-Mor, 2018 (link)). Endonucleases and DNA polymerase were purchased from New England Biolabs (Ipswich, MA, United States). The DNA purification kit, oligonucleotide synthesis, and DNA sequencing were provided by Sangon Biotech (Shanghai, China). To construct the fluorescent reporter plasmid pDH116, both the promoter of the ompA gene and encoding region of enhanced green fluorescent protein (EGFP) were amplified by primers in Supplementary Table 3. The two DNA fragments were purified and combined to a fusion by overlap extension PCR and were subsequently inserted into the backbone of plasmid pDH113 (Liu et al., 2015 (link)).

The strains used in this study are listed in Additional file 1: Table S1. EC135 lacking all R-M systems and orphan MTases was used as a cloning host to construct plasmids [40 (link)]. All strains were cultured in Luria–Bertani (LB) medium (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl) supplemented with the appropriate antibiotics (100 µg/mL ampicillin was used in E. coli and 20 µg/mL erythromycin, 10 µg/mL kanamycin, and 10 µg/mL chloromycetin were used in B. subtilis).
Kits for DNA purification/gel recovery and extracting genomic DNA, plasmid DNA, and RNA were purchased from TIANGEN Biotech (Beijing, China). DNA polymerase, restriction enzymes, and dNTPs were purchased from New England Biolabs (USA). Antibiotics, inducers, and standard chemicals, such as ampicillin, kanamycin, chloromycetin, erythromycin, IPTG, xylose, purine bases, nucleotides, and nucleosides were purchased from Sigma-Aldrich (USA). Tryptone and yeast extract were purchased from Oxoid Company (UK). Other reagents for cell culture and fermentation medium were all analytical pure, and purchased from Beijing Modern Oriental Fine Chemical Co., Ltd (China).

Kits for genomic DNA isolation, DNA purification and plasmid isolation were purchased from Omega (Norcross, GA, USA). E. coli DH5α and the pMD 18-T vector (TaKaRa, Otsu, Japan) were used for gene cloning and sequencing, respectively. Restriction endonucleases, T4 DNA ligase, DNA polymerase and dNTPs were purchased from New England Biolabs (Ipswich, MA, USA). Vector pET-28a(+) (Novagen, San Diego, CA, USA) and E. coli BL21 (DE3) (TaKaRa) were used for gene expression. Nickel-NTA agarose (Qiagen, Valencia, CA, USA) was used to purify the His6-tagged protein. The substrates pNP acetate (C2), pNP butyrate (C4), pNP caproate (C6), pNP caprylate (C8), pNP caprate (C10), pNP myristate (C14), pNP palmitate (C16) were purchased from Sigma (St. Louis, MO, USA). Isopropyl-β-D-1-thiogalactopyranoside (IPTG) was purchased from Amresco (Solon, OH, USA). All other chemicals were of analytical grade and commercially available.

Streptococcus agalactiae FPrA02^{45 (link)} was kindly provided by Dr. Channarong Rodkhum of the Department of Veterinary Microbiology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok. E. coli DH10 beta, restriction enzymes, DNA ligase, DNA polymerase and dNTPs were products of New England Biolabs Inc. (England). Quick-change kit was purchased from Stratagene (USA). pET-28a was from Novagen (USA). Plasmid purification kit was from Geneaid (Taiwan). HisTrap FF affinity column was from GE Healthcare (England). Standard oligosaccharides (G1–G7) and LR-CD were products of Wako Pure Chemical Industry Ltd. and Ezaki Glico (Japan), respectively. Glucose oxidase kit was from Human (Germany). Pea starch (Emsland-Starke GmbH, Germany) was kindly provided by Prof. Wolfgang Zimmermann of the University of Leipzig, Germany. All chemicals used were of analytical grade.

All chemicals are of analytical purity, purchased from Sigma-Aldrich (Australia) or Chem-Supply (Australia). Yeast extract and tryptone were purchased from Merck and Oxoid (Australia). Restriction enzymes, T4 ligase, and DNA polymerase were purchased from New England Biolabs (Australia) and were used according to the supplier’s recommendation. Plasmid isolations or DNA fragment purifications were done using GeneJET kits for Plasmid Miniprep, Gel Extraction, or the PCR Purification (Thermo Fisher Scientific Australia Pty. Ltd.).

Oligonucleotides were purchased from IDT, Singapore and the DNA polymerase, dNTPs (dATP, dGTP, dTTP, dCTP) Phusion buffer, restriction endonuclease (Sac I and Hind III), and T4 DNA ligase were purchased from NEB, USA. Pfu polymerase was purchased from Promega, USA. l-arabinose, protease inhibitor, lysozyme, Octyl β-D-glucopyranoside, nitrocellulose membrane, and primary antibody (anti-mouse monoclonal antibody) were purchased from Sigma-Aldrich, USA. Ni-NTA resin was procured from Qiagen. Ampicillin and kanamycin were purchased from HiMedia. Tris and phosphate buffer were purchased from MP Biomedical, LLC (France).