TY - JOUR
T1 - Factors involved in the electroporation-induced transformation of Clostridium perfringens
AU - Allen, Steven P.
AU - Blaschek, Hans P.
N1 - Funding Information:
This work was supported in part by New Investigator Research Award PHS 5 R23 A122417 from the National Institutes of Health to H.P.B., by Hatch grant 50-304 from the University of Illinois Agricultural Experiment Station, aa~l by grant 1-2-69157 from the University of Illinois Research Board. We thank Francis Macrina for plasmids pVA1 and pVA677, and Julian Rood for C. perfringens strain 13.
PY - 1990/7
Y1 - 1990/7
N2 - 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.
AB - 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.
KW - Clostridium perfringens 3624A Rif Str
KW - Electrotransformation
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U2 - 10.1016/S0378-1097(05)80042-4
DO - 10.1016/S0378-1097(05)80042-4
M3 - Article
C2 - 2227358
AN - SCOPUS:0025457533
VL - 70
SP - 217
EP - 220
JO - FEMS Microbiology Letters
JF - FEMS Microbiology Letters
SN - 0378-1097
IS - 2
ER -