Bacterial strains, plasmids, and primers used in this work are listed in Table 6 . C. perfringens NCTC 8237, NCTC 13110, and NCTC 8084 were used as donors of DNA. C. perfringens isolates were grown overnight under anaerobic conditions in Schaedler Anaerobe Broth and BBL Fluid Thioglycolate Medium at 37 °C.
Escherichia coli XL-1 Blue and NiCo21(DE3) (New England BioLabs, Ipswich, MA, USA) were used for DNA manipulation and toxins production, respectively. E. coli strains were grown at 37 °C aerobically on Luria-Bertani agar plates or in Luria-Bertani (LB) broth (Sigma Aldrich, St. Louis, MS, USA). Inducible production of proteins in E. coli NiCo21(DE3) was carried out in Terrific Broth (Merck) under shaking at 200 rpm. Kanamycin (50 µg/mL) was added to the medium. Enzymes for DNA cloning were supplied by New England BioLabs (NEB). Unless otherwise stated, all other reagents were from SERVA, Qiagen, or TaKaRa. All oligonucleotide primers were synthesized by Generi Biotech (Hradec Králové, Czech Republic). General genetic techniques for Clostridium and E. coli have been described previously [26 (link),27 ].
Escherichia coli XL-1 Blue and NiCo21(DE3) (New England BioLabs, Ipswich, MA, USA) were used for DNA manipulation and toxins production, respectively. E. coli strains were grown at 37 °C aerobically on Luria-Bertani agar plates or in Luria-Bertani (LB) broth (Sigma Aldrich, St. Louis, MS, USA). Inducible production of proteins in E. coli NiCo21(DE3) was carried out in Terrific Broth (Merck) under shaking at 200 rpm. Kanamycin (50 µg/mL) was added to the medium. Enzymes for DNA cloning were supplied by New England BioLabs (NEB). Unless otherwise stated, all other reagents were from SERVA, Qiagen, or TaKaRa. All oligonucleotide primers were synthesized by Generi Biotech (Hradec Králové, Czech Republic). General genetic techniques for Clostridium and E. coli have been described previously [26 (link),27 ].