Allen S P, Blaschek H P
Department of Food Science, University of Illinois, Urbana.
FEMS Microbiol Lett. 1990 Jul;58(2):217-20. doi: 10.1111/j.1574-6968.1990.tb13981.x.
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 microgram/ml concentration of transforming DNA, (3) use of late-logarithmic phase cells, (4) 3-fold concentration of cell density (3.0 x 10(8) 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/microgram DNA for plasmic pIP401 to 9.2 x 10(4) transformants per microgram DNA for plasmid pAK201. The greatest transformation efficiency obtained using pAK201 was 9.8 x 10(6) transformants/micrograms DNA for C. perfringens strain 13. Using the improved protocol, pAM beta 1 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.
已发现以下因素可提高产气荚膜梭菌3624A Rifr Strr的转化效率：(1) 将比色皿样品体积（DNA和细胞悬液）降至0.8 ml；(2) 使用浓度为1微克/毫升的转化DNA；(3) 使用对数后期的细胞；(4) 将细胞密度提高3倍（3.0×10⁸CFU/ml）；(5) 将表达和选择性平板培养基的pH值降至6.4。应用改进后的条件，产气荚膜梭菌3624A Rifr Strr的转化效率范围为：对于质粒pIP401，每微克DNA有7.1个转化子；对于质粒pAK201，每微克DNA有9.2×10⁴个转化子。使用pAK201获得的最高转化效率是产气荚膜梭菌13菌株每微克DNA有9.8×10⁶个转化子。与之前报道的值相比，使用改进后的方案，pAMβ1的转化水平提高了42倍[1]。除产气荚膜梭菌3624A Rifr Strr外，13、10543A、3628C、NTG - 4和3624A菌株也成功实现了转化。核酸酶似乎不是产气荚膜梭菌菌株特异性电转化方案中的一个因素。
