超耻垢分支杆菌基因敲除交流.ppt

耻垢分枝杆菌基因敲除交流 内容 sacB 作为负筛标记 sacB , 编码蔗糖6-果糖基转移酶 催化蔗糖水解并合成高分子量的果聚糖 在存在10%蔗糖时，sacB 基因的表达对耻垢分枝杆菌是致死的 其毒性的分子机制还不是很清楚 Pelicic V, Reyrat JM, Gicquel B. Expression of the Bacillus subtilis sacB gene confers sucrose sensitivity on mycobacteria. J Bacteriol 1996;178:1197-9. 构建自杀质粒 构建自杀质粒 1165 bp 6359 bp 5409 bp pMind-2630UD-sacB digests with Pac I and Spe I 电转化 200 ml 感受态细胞 5-10 ml 质粒(50–500 ng) 0.2 cm 电转杯2.5 kV , 5ms 37 ?C复苏4h 涂布相应平板 培养至OD 0.9 (600 nm) (冰浴 1.5 h)，4?C 3,000 rpm离心收集菌体 10%冰浴甘油洗涤两次， 4?C 3,000 rpm 离心 1/20th 原始培养基体积的冰浴 10% 甘油 200 μl分装-80 ?C保存 准备电转化感受态 电转化条件 一次双交换 Selective media were Luria-Bertani (or 7H10) medium with 10% sucrose , X-Gal and 50 mg/ml hygromycin 一步筛选带标记基因突变株 Selective media were Luria-Bertani (or 7H10) medium with X-Gal , 50 mg/ml kanamycin and 50 mg/ml hygromycin 两步筛选带标记基因突变株 第一次单交换 Selective media were Luria-Bertani (or 7H10) medium with 10% sucrose , X-Gal and 50 mg/ml hygromycin, confirmed by testing kanamycin sensitivity 第二次单交换 两步筛选无标记基因突变株 确认敲除菌株 其他说明 * * * * * * * * * *