A simple procedure for genetic transformation of Streptococcus sanguis Challis was developed and standardized. During the exponential phase of growth, cells became competent while growing as diplococci in broth con- taining 10 % foetal calf serum. High levels of competence were maintained by the cultures for 60 min. Competent cells could be stored frozen without loss of competence for at least three years. Using total chromosomal DNA as donor, the dose-response curve for transformation of a point mutation (streptomycin resistance) showed one-hit kinetics, as the DNA concentration varied from 0.000001 to 10 ~tg/ml. At 10 ~tg/ml, more than 2.2 % of the colony-forming units were transformed to streptomycin resistance, while trans- forming activity remained detectable with 1 pg of DNA/ml. Optimal time of exposure of competent cells to transforming DNA was 30 min. The trans- formation reaction was inhibited at 0 and 4°C, whereas it occurred efficient- ly both at 25 and 37°C.
Method and parameters for genetic transformation of Streptococcus sanguis Challis.
LIEVENS, Patricia;
1990-01-01
Abstract
A simple procedure for genetic transformation of Streptococcus sanguis Challis was developed and standardized. During the exponential phase of growth, cells became competent while growing as diplococci in broth con- taining 10 % foetal calf serum. High levels of competence were maintained by the cultures for 60 min. Competent cells could be stored frozen without loss of competence for at least three years. Using total chromosomal DNA as donor, the dose-response curve for transformation of a point mutation (streptomycin resistance) showed one-hit kinetics, as the DNA concentration varied from 0.000001 to 10 ~tg/ml. At 10 ~tg/ml, more than 2.2 % of the colony-forming units were transformed to streptomycin resistance, while trans- forming activity remained detectable with 1 pg of DNA/ml. Optimal time of exposure of competent cells to transforming DNA was 30 min. The trans- formation reaction was inhibited at 0 and 4°C, whereas it occurred efficient- ly both at 25 and 37°C.
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