Open AccessElectrotransformation of Streptococcus agalactiae with plasmid DNA
Author(s) -
Ricci M.Luisa,
Manganelli Riccardo,
Berneri Cesare,
Orefici Graziella,
Pozzi Gianni
Publication year - 1994
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.1994.tb06865.x
Subject(s) - streptococcus agalactiae , plasmid , microbiology and biotechnology , streptococcus , biology , dna , streptococcaceae , bacteria , genetics , antibiotics
A protocol for efficient electrotransformation of Streptococcus agalactiae (group B streptococcus) Lancefield's strain O90R (NTCT 9993) (an unencapsulated derivative of type Ia strain O90) was developed. The Escherichia coli ‐ Streptococcus shuttle vector pDP28 (7.8 kb) carrying the ermB gene for resistance to erythromycin was used as donor DNA. Frozen ‘electrocompetent’ cells were prepared by repeated washes in 10% glycerol. A 50‐μl aliquot containing about 5×10 9 colony forming units of bacteria was subjected to the electric pulse. Optimal conditions for electrotransformation were determined using different media, harvesting cells at different points of the growth curve, and using different field strengths. The dose‐response curve for transformation of S. agalactiae with pDP28 showed one‐hit kinetics as donor DNA varied between 0.01 and 3 μg. The efficiency of electrotransformation for this range of amounts of donor DNA was 1.2×10 4 cfu μg −1 . The transformation frequencies obtained with this electroporation protocol are high enough to allow both subcloning and shotgun cloning of streptococcal DNA in S. agalactiae .