E coli dh5α

T4 DNA ligase, KOD-Plus-Neo polymerase, EcoRI, NotI, and SacI were purchased from Bao Bioengineering, Co., Ltd. (Dalian, China). PMD18-T vector, pPIC9K vector, and E. coli DH5α were purchased from Shanghai Sangon Biotech Co., Ltd. (Shanghai, China). Minimal dextrose (MD), buffered glycerol-complex (BMGY), and buffered methanol-complex (BMMY) medium were prepared according to the recipe provided in the Invitrogen Pichia pastoris expression kit instruction manual.

E. coli DH5α (Sangon Biotech, Shanghai, China) and B. subtilis WB800 (preserved in our laboratory) were hosts for cloning and expression, respectively. The shuttle plasmid pMA5 with NdeI and BamHI sites (Talen Biotech, Shanghai, China) was used as the expression vector. Luria-Bertani (LB) broth (5 g/L yeast extract, 10 g/L tryptone, and 10 g/L NaCl) was prepared for the routine growth of E. coli. Ampicillin was dissolved in sterile water and used at a final concentration of 100 μg/mL. A super-rich medium (SR) containing 50 μg/mL kanamycin consisted of 3 g/L K₂HPO₄, 20 g/L yeast extract, 25 g/L tryptone, and 30 g/L glucose for B. subtilis WB800 cultivation.

Six liver tissues were cut into pieces and snap-frozen for homogenization to extract total liver RNA using the Total RNA Extraction Kit for Animal Tissues (Foregene, Chengdu, China). The concentration and purity of RNA were determined using a NanoDrop-1000 Spectrophotometer (Thermo Scientific, Wilmington, DE, USA). A single total RNA ≥ 1 µg, concentration ≥ 35 ng/µL, OD 260/280 ≥ 1.8, and OD 260/230 ≥ 1.0. Then, cDNA was synthesized from 1 µg of total RNA by using the PrimeScript™ RT Reagent kit (Takara, Dalian, China). Primer Premier 6.0 was used to design primers to amplify cDNA sequences based on our full-length transcripts of S. prenanti [54 (link)]. The PCR products were purified using a PCR purification kit (TianGen kit, China) and ligated to the pMD19-T vector. Then, construct was transformed into the E. coli DH5α and the sequence was verified by Sanger sequencing (Sangon Biotech, Shanghai, China). Open reading frames (ORFs) were predicted using the ORF Finder tool (https://www.ncbi.nlm.nih.gov/orfnder/ (accessed on 25 July 2023)). The conserved domains were predicted using the NCBI Conserved Domain Database (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi/ (accessed on 28 July 2023)). Multiple sequences were aligned and phylogenetic analysis were performed using MEGA 6.0 software.

E. coli DH5α (Shanghai Sangon Biological Engineering Co. Ltd., Shanghai, China) was used for plasmid amplification and P. pastoris X-33 was used for the expression of the fusion protein. The pPICZα-A expression vector and P. pastoris X-33 were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). The restriction endonuclease, T4 DNA ligase and Taq DNA polymerase were purchased from Takara Biotechnology Co., Ltd. (Dalian, China). Ni-chelating Sepharose columns were purchased from Shanghai Sangon Biological Engineering Technology and Services Co., Ltd. (Shanghai, China).

V. parahaemolyticus 17802, Salmonella typhimurium 14028 and Staphylococcus aureus 43300 were purchased from the American Type Culture Collection (ATCC). V. parahaemolyticus isolates VP 1.1, VP 1.3, VP 2.2, VP 3.1, VP 3.2, VP 4.1, VP 304, VP 461, VP 800 and VP l4-90, Vibrio harveyi V1, Shewanella baltica S1, Acinetobacter baumannii A1, Pseudomonas fluorescens P1, Aeromonas hydrophila A1, Pseudomonas putrefaciens P1, Vibrio anguillarum V1 were stored in the Food Safety Lab at the Ocean University of China. E. coli DH5α and E. coli BL21 (DE3) were purchased from Sangon Biotech (Shanghai, China). All marine bacterial species were routinely cultured in the Zobell 2216 marine medium (Hopebio, Qingdao, China), whereas the other bacteria were grown in LB medium (Hopebio, Qingdao, China). Working concentrations of antibiotics were added into the growth medium when necessary.

The complete coding region of BrGOLDEN was amplified from the cDNA by PCR using a pair of primers with a homologous arm (Table S4). The PCR product was inserted into the pCAMBIA1305 vector digested with BamH I and Xba I using a Trelief SoSoo Cloning Kit (TSINGKE, Beijing, China) to generate pCAMBIA1305−BrGOLDEN_Ins, pCAMBIA1305−BrGOLDEN_Ldel, and pCAMBIA1305−BrGOLDEN_Del. The recombinant plasmid was then transferred into E. coli DH5α and sent to Sangon Biotechnology for sequencing to verify the correct insertion. We subsequently introduced the overexpression construct into Agrobacterium tumefaciens strain GV3101. pCAMBIA1305−BrGOLDEN_Ins, pCAMBIA1305−BrGOLDEN_Ldel, and pCAMBIA1305−BrGOLDEN_Del were then mixed to transform A. thaliana [75 (link)].

The Escherichia coli DH5α (Invitrogen, USA) was grown in an LB (Luria-Bertani) medium (Difco) at 37°C for cloning and the construction of plasmids. The Lactococcus lactis subsp. lactis CICC 6242 strain used in the experiments was supplied by the China Center of Industrial Culture Collection (Beijing, People’s Republic of China). The pGEM-T Easy Vector (Promega, Madison, USA) and pMG36e vector (Addgene, MA, USA) were used in the cloning and recombinant expression experiments. The different fermentations of L. lactis subsp. lactis were done in 250-mL flasks containing 50 mL of M17 medium (Difco) containing 0.5% of glucose. The L. lactis subsp. lactis was incubated by shaking at 100 rpm at 30°C for 18 hours without control of pH. The erythromycin (Sangon, Shanghai, China) was used at 250 μg/mL for E. coli DH5α and 5 μg/mL for L. lactis subsp. lactis strains, respectively. The cell growth (O.D. 600), pH and nisin titers (IU/ml) parameters were evaluated every two hours until 18 hours of fermentation.