FACTORS INVOLVED IN THE ELECTROPORATION-INDUCED TRANSFORMATION OF CLOSTRIDIUM-PERFRINGENS

The following factors were found to improve the efficiency of transformation of Clostridium perfringens 3624A Rifr Strr: (1) a reduction in cuvette sample volume (DNA and cell suspension) to 0.8 ml, (2) use of a 1 g/ml concentration of transforming DNA, (3) use of late-logarithmic phase cells, (4) 3-fold concentration of cell density (3.0 × 108 CFU/ml), and (5) a reduction in the pH of the expression and selective plating medium to 6.4. Application of the improved conditions resulted in transformation efficiencies for C. perfringens 3624A Rifr Strr ranging from 7.1 transformants/g DNA for plasmic pIP401 to 9.2 × 104 transformants per g DNA for plasmid pAK201. The greatest transformation efficiency obtained using pAK201 was 9.8 × 106 transformants/g DNA for C. perfringens strain 13. Using the improved protocol, pAMβ1 was transformed at a 42-fold greater level when compared with the values reported earlier [1]. In addition to C. perfringens 3624A Rifr Strr, strains 13, 10543A, 3628C, NTG-4, and 3624A were successfully transformed. Nuclease does not appear to be a factor in the C. perfringens strain-specific electro-transformation protocol. © 1990 Federation of European Microbiological Societies.