产品介绍
pHT01枯草胞内质粒
基本信息
启动子: Lac
复制子: ColE1 ori
质粒分类: 广宿主系列,枯草杆菌载体
质粒大小: 7956bp
原核抗性: Amp
筛选标记: Chl
克隆菌株: DH5α
培养条件: 37℃,有氧 LB
表达宿主: 枯草芽孢杆菌
诱导方式: IPTG诱导
5'测序引物: 根据序列设计引物
3'测序引物: 根据序列设计引物
质粒简介
All vectors use the strong promoter preceding the groESL operon of Bacillus subtilis fused to the lac operator allowing their induction by addition of IPTG. While the background level of expression of these expression cassettes is very low in the absence of the inducer, an induction factor of about 1,300 was measured using the bgaB reporter gene. The amount of recombinant protein produced after addition of IPTG may represent 10 and 13%, respectively, of the total cellular protein. High level secretion of amyQ α-amylase and cellulase A and B of Clostridium thermocellum was demonstrated. An efficient Shine-Dalgarno sequence as well as a multiple cloning site (BamH I, Xba I, AatII, SmaI) were also inserted. To obtain secretion of recombinant proteins, the coding region for the signal peptide of the amyQ gene encoding an α-amylase was fused to the SD sequence of pHT01, thereby constructing pHT43.
大肠杆菌-枯草芽孢杆菌穿梭质粒的表达载体pHT01可以在枯草芽孢杆菌中高效表达重组外源蛋白。载体基于强σA-依赖性启动子的枯草杆菌groE操纵子,通过添加lac操纵子改造成为一种高效可控的(IPTG诱导的)启动子。
质粒图谱
pHT01枯草胞内质粒使用说明:
1、收到质粒干粉后请先5000rpm离心1min,再加入20μl无菌水溶解质粒,室温放置1min;
2、从-80℃冰箱中取出相应的感受态,置于冰盒上解冻,并做好标记;
3、取2μl质粒加至100μl感受态中,冰浴30min;
4、42℃热激90s,再冰浴2min;
5、加入900μl无抗的LB液体培养基,180rpm震荡培养45min;
6、6000rpm离心5min,仅留100ul上清混匀菌体沉淀;
7、混匀后的菌液加至对应抗性的LB平板上,倒入适量玻璃珠,涂匀液体;
8、将平板正向培养1h,再倒置培养12h~16h;
9、挑取单克隆菌落至对应抗性的LB液体培养基中,震荡培养12h~16h,根据实验需要提取质粒。
pHT01枯草胞内质粒注意事项:
1、如果您收到的是甘油菌种,请先四区划线,挑取单克隆培养。
2、如果第二天转化平板长的过多,请将质粒按比例稀释后再转化。