Restriction endonuclease

Plasmid DNA was prepared using the Monarch^® Plasmid Miniprep Kit according to the manufacturer’s instructions (NEB). Enzymes for DNA manipulations included restriction endonucleases (NEB), T4 DNA ligase (NEB) and Calf Intestinal Alkaline Phosphatase (Invitrogen™, Thermo Fisher Scientific, Loughborough, UK). Gene cleaning was performed using the Monarch^® PCR & DNA Cleanup Kit (NEB) following the manufacturer’s instructions. DNA polymerase used for PCRs was Q5^® High-Fidelity DNA Polymerase (NEB) for cloning purposes or Taq DNA polymerase (NEB) for verification of constructs or for colony PCR. PCRs were performed in an Eppendorf^® Mastercycler^® (Stevenage, UK)). Reaction mixtures were typically subjected to initial denaturation at 94 °C for 5 min followed by 35 cycles: Denaturation at 94 °C for 30 s, annealing at the appropriate temperature for the primers for 30 s and extension at 72 °C for 30 s to 1 min depending on the length of amplicon, and a final extension cycle at 72 °C for 5 min. The amplified DNA was visualised by electrophoresis with 1% w/v agarose gels.

Restriction endonucleases and T4 DNA ligase were from Thermo Fisher Scientific (Waltham, MA, USA). Oligonucleotides were synthesized in-house from commercially available phosphoramidites (Glen Research, Sterling, VA, USA). The sequences are listed in Table 2. Oligonucleotides were 5′-labeled using γ[³²P]-ATP (ICBFM Laboratory of Biotechnology, Novosibirsk, Russia) and T4 polynucleotide kinase (Biosan, Novosibirsk, Russia). Inhibitors were purchased from Vitas-M Chemical Ltd. (Hong Kong, China).

The bacterial strains and plasmids used in this study are listed in Table S1. Escherichia coli DH5α was used as a host for cloning vectors. B. subtilis SCK6 was the original strain used in the genome engineering procedures. Plasmids and genomic DNA were extracted using the kit (TIANGEN) according to the manufacturer’s instructions. PCR used the polymerase Taq (Vazyme). Restriction endonucleases and T4 DNA ligase were purchased from Thermo Scientific. During strain construction, the culture was grown aerobically in Luria-Bertani (LB) liquid medium (1% [wt/vol] trptone, 0.5% [wt/vol] yeast extract, 1% [wt/vol]) NaCl and LB agar plates at 30 or 37°C. Ampicillin (100 mg/liter), kanamycin (50 mg/liter), or spectinomycin (50 mg/liter) were used to select suitable strains. l-Arabinose was added for induction at a concentration of 1%, and the isopropyl-β-d-thiogalactoside (IPTG) was used at concentration of 0.5 mmol/liter.

The strains used for antimicrobial activity determination, E. coli ATCC 25922, E. coli ATCC 078, E. coli UB 1005, Salmonella Typhimurium (S. Typhimurium) C 7731, S. Typhimurium ATCC 14028, P. aeruginosa ATCC 27853, Staphylococcus aureus (S. aureus) ATCC 29213, S. aureus ATCC 25923, Staphylococcus epidermidis (S. epidermidis) ATCC 12228, and Streptococcus faecalis (S. faecalis) ATCC 29212, were all preserved by the Institute of Animal Nutrition, Northeast Agricultural University. The vector pPICZaA was purchased from Liuhe Huada Gene Technology Co., Ltd. (Beijing, China). Restriction endonucleases were obtained from Thermo Fisher Co., Ltd. (Waltham, USA), and SUMO protease was purchased from Gene Copoeia (Guangzhou, China). A plasmid extraction kit was obtained from Genstar (Beijing, China). The Ni–NTA Sefinose (TM) resin kit was purchased from Sangon Biotech Co., Ltd. (Shanghai, China). The purity of other chemical reagents used was of analytical grade.

Dithiothreitol (DTT), Tris-base, phenyl methylsulfonyl fluoride (PMSF) and NaCl, were purchased from Sangon (Shanghai, China). KOD-plus mutagenesis kits were obtained from TOYOBO (Osaka, Japan). All restriction endonucleases, T4 DNA ligase, T4 Polynucleotide Kinase, Dynabeads protein G and Lipofectamine 2000 transfection reagent were obtained from Thermo Scientific (Waltham, MA, USA). All materials for the yeast two-hybrid screening and assay, including the Matchmaker Gold Yeast Two-Hybrid System, media, reagents, and a normalized Mate & Plate human cDNA library, were purchased from Clontech (Shiga, Japan). Escherichia coli Rosetta (DE3) cells were purchased from Stratagene (Santa Clara, CA, USA). Polyvinylidene fluoride (PVDF) membranes were obtained from Millipore (Darmstadt, Germany). DNA sequencing was performed by Biosune (Shanghai, China).