Kanamycin sulphate

All chemicals and biological materials were obtained from commercial suppliers. Lysozyme, DNase I, kanamycin sulphate and chloramphenicol were purchased from Sigma-Aldrich; polymyxin B sulfate from AlfaAesar; LB agar, LB medium, 2 × YT medium and arabinose from Formedium; Escherichia coli (E. coli) 5α, Q5 DNA polymerase, T4 DNA ligase and restriction enzymes from New England BioLabs; N_δ-methylhistidine (MeHis; H-His(3-Me)-OH) from Bachem; E. coli DH10B from Thermo Fisher; and oligonucleotides were synthesized by Integrated DNA Technologies.

The previously obtained C. pseudotuberculosis CP09 mutant strain [7 (link)], the T1 pathogenic wild-type parental strain [11 (link)], and the caprine-pathogenic strain MIC-6 from the Laboratory of Genetics and Control of Microorganisms (Belo Horizonte, Brazil) bacterial collection were employed in this work. Strains were aerobically grown in “Brain Heart Infusion” broth (BHI, Oxoid) at 37°C. Kanamycin (Kanamycin sulphate 25 μg/mL; Sigma-Aldrich, USA) was added to the mutant growth media. T1 and MIC-6 strains were isolated from caseous lesions of goats and have previously been employed in vaccination trials using goats and mice [11 (link),12 (link)].

E. coli BL21(DE3) harbouring the recombinant plasmid pD861-CBD-Ssp DnaB-hEGF [3070 bp]was used as a host for CBD-Ssp DnaB-hEGF expression. The recombinant plasmid was constructed by our team and synthesized by ATUM (Newmark, CA, USA). The CBD-Ssp DnaB-hEGF was successfully expressed by Yosua et al. (2021) as a 31.4 kDa protein. Ammonium persulphate, coomassie brilliant blue G-250, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reagent, sodium chloride, urea, Tris base, and β-mercaptoethanol were purchased from Merck (USA). Acrylamide/bis solution, molecular weight marker, and Tetramethylethylenediamine (TEMED) were purchased from Bio-Rad (USA). EDTA disodium salt was purchased from 1^st base (Singapore). Yeast extract was obtained from Oxoid (UK). Dialysis tubing (MWCO 3 kDa), kanamycin sulphate, L-rhamnose, recombinant hEGF, tryptone, tricine, and Triton X-100 were purchased from Sigma-Aldrich (Singapore). Oxidised (GSSG) and reduced glutathione (GSH) were purchased from Tokyo Chemical Industry (Japan). Anti-EGH monoclonal antibody and alkaline phosphatase-conjugated anti-mouse IgG antibody were purchased from Santa Cruz Biotechnology (USA). LB medium was prepared from 1% (w/v) sodium chloride, 1% (v/v) tryptone, and 0.5% (v/v) yeast extract.

Five types of apple tissue media were used in this study. These included the four media (shoot maintenance medium, shoot regeneration medium, rooting medium and MS20 medium) described by Yao et al. [4 (link)], and a leaf expansion medium (Table S3). All media were adjusted to pH 5.8 with NaOH and then autoclaved at 1.1 kg cm⁻² (121°C) for 15 min. For transformation experiments the antibiotics: cefotaxime (Claforan, Roussel NZ Ltd); kanamycin sulphate (Sigma); and the herbicide Glean (Du Pont, active agent chlorsulfuron) were filter-sterilized and added where appropriate to media after autoclaving. For maintaining shoot cultures and rooting, 290-mL plastic containers (Aimed TC290SP, 8 × 6 cm, diameter × height) each containing 50 mL medium were used to culture 8–10 shoots per container. For co-cultivation and shoot regeneration experiments, Petri dishes (8.5 cm diameter) each containing 20 mL medium were used to culture up to 15 explants per plate. The conditions of the plant tissue culture room were set at 24°C and 16-h photoperiod (30 μmol m⁻² s⁻¹).

n-Butanol was supplied by J.T. Baker (Xalostoc, México) and culture media (Difco®) by Becton Dickinson de México, S.A. de C.V. (CDMX, México). In addition, kanamycin sulphate, diosgenin 98%, and p-anisaldehyde 93% were purchased from Sigma-Aldrich (St. Louis, MO, USA, 2017). Methanol and antioxidant reagents DPPH 2,2-Diphenyl-1-picrylhydrazyl, potassium persulfate, ABTS 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, sodium acetate, TPTZ (2,4,6-Tris(2-pyridyl)-s-triazine), hydrochloric acid, fluorescein by Sigma-Aldrich, and 2,2′-Azobis(2-methylpropionamidine) dihydrochloride were purchased to Sigma-Aldrich (Toluca, Mexico). Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) Millipore was purchased from Merck (México).

Glucose, fructose, and sucrose were from Merck (Darmstadt, Germany). Melezitose, kanamycin sulphate, isopropyl-β-D-1-thiogalactopyranoside (IPTG), 3,5-dinitrosalicylic acid (DNS) and beef extract were from Sigma-Aldrich (Saint Lois, MO, USA). Polysorbate 80 was from Santa Cruz Biotechnology (Dallas, TX, USA) and 3-(N-morpholino)-propanesulfonic acid (MOPS) from NzyTech (Lisbon, Portugal). Sodium alginate Protanal LF 120 LS was from FMC Biopolymer (Grivan, Ayrshire, UK). Beghin Meiji SA (Neuilly-sur-Seine, France) kindly supplied the commercial fructo-oligosaccharides mixture “Actilight”. Yeast extract, peptone and tryptone were from Laboratorios Conda S.A. (Madrid, Spain).