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 Rif^r Str^r: (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 Rif^r Str^r 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 Rif^r Str^r, 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.