大肠杆菌毒素-抗毒素系统ccd(control of cell division or death system)编码的毒素蛋白CcdB使细胞内DNA促旋酶失活,杀伤宿主细胞,而抗毒素蛋白CcdA可以中和毒素CcdB使宿主存活。利用这个原理,CcdB可作为细菌转化时的筛选标记,在构建...大肠杆菌毒素-抗毒素系统ccd(control of cell division or death system)编码的毒素蛋白CcdB使细胞内DNA促旋酶失活,杀伤宿主细胞,而抗毒素蛋白CcdA可以中和毒素CcdB使宿主存活。利用这个原理,CcdB可作为细菌转化时的筛选标记,在构建各种高效低背景载体上发挥重要作用。我们简要综述毒素蛋白CcdB的毒性原理及其在质粒载体构建中的广泛应用。展开更多
Introducing PCR products into plasmids vectors is key for molecular techniques. Ideally cloning vectors are easy to construct, modify and propagate, neither require advanced techniques nor special equipment or reagent...Introducing PCR products into plasmids vectors is key for molecular techniques. Ideally cloning vectors are easy to construct, modify and propagate, neither require advanced techniques nor special equipment or reagents and efficiently incorporate PCR products at close to zero empty vector background. We provide an easy to engineer self-made cloning vector, neither requiring sophisticated tools or techniques nor advanced cloning knowledge. Through recombination we obtained the pUC18ccdB vector, carrying the ccdB suicide gene within the pUC18 backbone. When SmaI cleaved (within the ccdB) vector was T4 ligated with small (0.2 kbp) and intermediate (1.3 to 2.2 kbp) blunt end PCR-products and transformed into E. coli, the amount of clones with incorporated PCR product was comparable to commercial PCR-cloning kits and at a close to zero PCR product negative background. In conclusion we present a simple, versatile and cheap approach to an efficient “home made” PCR-cloning vector that allows integration of crude blunt end PCR products at close to zero background.展开更多
ccdAB系统(control of cell division or death system)是目前已知的一种毒素-抗毒素系统(toxin-antitoxin system,TA系统),存在于致病性大肠杆菌F质粒及染色体骨架上,由ccdA和ccdB两个基因组成。质粒上的ccdAB系统编码一种毒素蛋白CcdB...ccdAB系统(control of cell division or death system)是目前已知的一种毒素-抗毒素系统(toxin-antitoxin system,TA系统),存在于致病性大肠杆菌F质粒及染色体骨架上,由ccdA和ccdB两个基因组成。质粒上的ccdAB系统编码一种毒素蛋白CcdB,在缺乏抗毒素的情况下,CcdB使细胞内促旋酶中毒,从而干扰DNA的合成,杀伤宿主细胞。本文对ccdAB系统的结构和功能,以及所编码CcdB的作用机制进行了综述。展开更多
文摘大肠杆菌毒素-抗毒素系统ccd(control of cell division or death system)编码的毒素蛋白CcdB使细胞内DNA促旋酶失活,杀伤宿主细胞,而抗毒素蛋白CcdA可以中和毒素CcdB使宿主存活。利用这个原理,CcdB可作为细菌转化时的筛选标记,在构建各种高效低背景载体上发挥重要作用。我们简要综述毒素蛋白CcdB的毒性原理及其在质粒载体构建中的广泛应用。
文摘Introducing PCR products into plasmids vectors is key for molecular techniques. Ideally cloning vectors are easy to construct, modify and propagate, neither require advanced techniques nor special equipment or reagents and efficiently incorporate PCR products at close to zero empty vector background. We provide an easy to engineer self-made cloning vector, neither requiring sophisticated tools or techniques nor advanced cloning knowledge. Through recombination we obtained the pUC18ccdB vector, carrying the ccdB suicide gene within the pUC18 backbone. When SmaI cleaved (within the ccdB) vector was T4 ligated with small (0.2 kbp) and intermediate (1.3 to 2.2 kbp) blunt end PCR-products and transformed into E. coli, the amount of clones with incorporated PCR product was comparable to commercial PCR-cloning kits and at a close to zero PCR product negative background. In conclusion we present a simple, versatile and cheap approach to an efficient “home made” PCR-cloning vector that allows integration of crude blunt end PCR products at close to zero background.
文摘ccdAB系统(control of cell division or death system)是目前已知的一种毒素-抗毒素系统(toxin-antitoxin system,TA系统),存在于致病性大肠杆菌F质粒及染色体骨架上,由ccdA和ccdB两个基因组成。质粒上的ccdAB系统编码一种毒素蛋白CcdB,在缺乏抗毒素的情况下,CcdB使细胞内促旋酶中毒,从而干扰DNA的合成,杀伤宿主细胞。本文对ccdAB系统的结构和功能,以及所编码CcdB的作用机制进行了综述。
