Electrotransformation of Streptococcus agalactiae with plasmid DNA |
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Authors: | MLuisa Ricci Riccardo Manganelli Cesare Berneri Graziella Orefici Gianni Pozzi |
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Institution: | Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Roma, Italy; Sezione di Microbiologia, Dipartimento di Biologia Molecolare, Universitàdi Siena, Via Laterina 8, 53100 Siena, Italy; Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia, Via A. Bianchi 7, 25100 Brescia, Italy |
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Abstract: | Abstract 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 5×109 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 1.2×104 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 . |
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Keywords: | Streptococcus agalactiae Electrotransformation Shuttle vector |
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