Improved electroporation and cloning vector system for gram-positive bacteria.
AUTOR(ES)
Dunny, G M
RESUMO
A protocol for transformation of intact Enterococcus faecalis cells by electroporation was developed through a systematic examination of the effects of changes in various parameters, including (i) growth conditions; (ii) composition of the electroporation solution; (iii) electroporation conditions, such as field strength and resistance; (iv) size, concentration, and purity of DNA used for transformation; and (v) conditions used to select for transformants. Key features of this protocol include the use of exponential-phase cells grown in inhibitory concentrations of glycine and the use of an acidic sucrose electroporation solution. Frequencies of greater than 2 x 10(5) transformants per microgram of plasmid DNA were obtained for E. faecalis cells, whereas various strains of streptococci and Bacillus anthracis were transformed at frequencies of 10(3) to 10(4) transformants per microgram of plasmid DNA with the same protocol. A novel Escherichia coli-Streptococcus and Enterococcus shuttle cloning vector, pDL276, was constructed for use in conjunction with the electroporation system. This vector features a multiple cloning site region flanked by E. coli transcription termination sequences, a relatively small size (less than 7 kb), and a kanamycin resistance determinant expressed in both gram-positive and gram-negative hosts. Various enterococcal and streptococcal DNA sequences were cloned in E. coli (including sequences that could not be cloned on other vectors) and were returned to the original host by electroporation. The vector and electroporation system was also used to clone directly into E. faecalis.
