Electrotransformation of streptococcus with lacotococci plasmid and investigation of the factor of dental caries by oral microorganisms

1. 从乳酸乳球菌JK301pGB301中提取pGB301，采用碱- sds -超离心法提取pGB301。琼脂糖凝胶电泳结果显示为单条带。细粒链球菌10707、血链球菌ATCC 35105和gordonii chis的电转化及其效率将这三种微生物菌株培养在添加5%马血清的Todd-Hewitt肉汤中。微生物通过转化在红霉素培养皿上生长。其效率范围为1.5×10^4到5.5×10^4。电穿孔的效率提高了5倍。当微生物的光密度为肉汤光密度的0.1时收获效果最好。密氏霉10707低ph敏感突变体的构建。以甲基磺胺乙酯为原料，构建了米氏霉10707低ph敏感突变体。亲本菌株在pH 4.2的培养液中生长。另一方面，突变体在pH为4.6的培养液中不生长。转化效率为1.4 ×10^3 CFU/μg.4。当峰值电压为2.5 kV，电容为25 F，脉冲控制为200Ω，收获OD为0.5.5时，获得了最有效的电转化条件。口腔微生物对β-半乳糖糖的致龋因子和变形链球菌所涉及的酶的活性不利用β-半乳糖糖和乳糖果糖。

1. Extraction pGB301 from Lactococcus lactis JK301pGB301 was extracted by means of alkali-SDS and ultracentrifusion. A single band was confirmed on agarosen gel electrophoresis.2. Electrotransformation of Streptococcus milleli 10707, S.sanguis ATCC 35105 and S.gordonii Challis and its efficiencyThe strains of the microorganisms were cultured in Todd-Hewitt broth sulpplemented with 5% horse serum. The microorganisms grew on plate with Erythromycin by the transformation. Its efficiency was ranged form 1.5×10^4 to 5.5×10^4. The effciency increased five times by electroporation. It was most effective whhen the microorganisms were harvested at 0.1 of optical density of the broth.3. Construction of low-pH sensitve mutansts of S.milleli 10707.Low-pH sensitve mutansts of S.milleli 10707 were constructed with ethyl methasulfonete. The parent strain grew in the broth of pH 4.2. On the other hand the mutant did not grow in the broth of pH 4.6. Its transformation efficiency was 1.4 ×10^3 CFU/μg.4. The most effective condition for electrotransformationThe most effective condition for electrotransformation was obtained when peak voltage was 2.5 kV, capacitance was 25 F, pulse controll was 200Ω and harvest OD was 0.5.5. Cariogenic factos of β-galacto-sugars by oral microorganisms and the activity of the enzymes involvedMutans streptococci did not utilize β-galactosyllactose and lactosylfructoside.