ccdB基因位于大肠杆菌的 F质粒 上，是被ccd操纵子编码的毒性-抗毒性系统的一部分，确保含F质粒的细胞分裂后F质粒仍存在（分裂后含F质粒的细胞才能存活）。 ccdB编码的毒性蛋白 CcdB，作为DNA促旋酶抑制剂，锁定DNA促旋酶和断裂的双链DNA复合物，使DNA促旋酶不能发挥作用，最终导致细胞死亡。 ccdA是ccd操纵子中的另一个基因，编码抗毒性蛋白 CcdA，保护细胞免受CcdB的毒性作用。 细胞因为丢失F质粒而丢失了ccdA基因，导致细胞屈服于CcdB的毒性。 F …

2016年6月2日 · The BAC vector is generated by inserting into pBAC2015 a ccdB gene together with an ampicillin resistance gene and a pBR322 replication origin flanked by homology arms and unique restriction ...

2023年1月1日 · In this study, we demonstrated a streamlined approach for creating infectious BAC clones of PRV by ExoCET. The linear BAC vector flanking the homologous sequences was released from an intermediate plasmid containing a pBR322 replication origin and the ccdB gene. The high-copy replicon ensured good yield and high DNA quality for production of ...

2013年12月24日 · Seamless BAC mutagenesis using ccdB counterselection. A functional test based on β-galactosidase expression from pBeloBAC11 was used to evaluate the efficiency and fidelity of ccdB counterselection for BAC recombineering (Figure 4A). The lacZ gene of pBeloBAC11 was first disrupted with the ccdB-amp cassette.

2010年12月13日 · Recombineering, permitting precise modification of genes within bacterial artificial chromosomes (BACs) through homologous recombination mediated by lambda phage-encoded Red proteins, is a widely used powerful tool in …

2024年7月15日 · By combining recombineering, ccdB counterselection and exonuclease-mediated in vitro annealing, RedEx achieves seamless mutagenesis of large DNA molecules, including plasmids, fosmids and BACs.

2023年8月1日 · The ccdB gene, which codes for the toxic protein CcdB, is commonly used for counter-selection systems, including BAC recombineering [11]. Expression of CcdB is toxic to E. coli in the absence of the ccdA gene product, which is the antitoxin protein CcdA .

2022年5月19日 · Here we describe two recombineering protocols for modification and transfer of plant genes from BACs into Agrobacterium T-DNA plant transformation vectors. The first protocol uses a conditional suicide ccdB gene cassette to assist the genetic complementation assays by generation of point mutations, deletions, and insertions at any gene position.

基因组文库构建耗时费力，而且 柯斯质粒 （Cosmid）文库无法克隆超过45kb的片段， 细菌人工染色体 （BAC）文库虽然可以获得大于200kb的基因组DNA，但是只有专业实验室才能实现。直接克隆技术可以将基因组中的DNA大片段直接捕获到载体上，避免了繁琐的DNA文库 ...

Gateway 的核心之一就是利用 ccdB 的毒性，从而达到negative selection. ccdB是一种DNA gyrase， 其工作原理如下图： 它能让DNA的超螺旋结构变成逆向螺旋，因此对 E.coli 具有毒性。 Gateway如何利用它的毒性呢？