Improved electroporation and cloning vector system for gram-positive bacteria

G. M. Dunny, L. N. Lee, D. J. LeBlanc

Research output: Contribution to journalArticlepeer-review

248 Scopus citations

Abstract

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 >2 x 105 transformants per μg of plasmid DNA were obtained for E. faecalis cells, whereas various strains of streptococci and Bacillus anthracis were transformed at frequencies of 103 to 104 transformants per μg 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 (<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.

Original languageEnglish (US)
Pages (from-to)1194-1201
Number of pages8
JournalApplied and environmental microbiology
Volume57
Issue number4
DOIs
StatePublished - 1991

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