Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand.
Next Generation Genomic Co. Ltd., Lumpini, Pathumwan, Bangkok, 10330, Thailand.
World J Microbiol Biotechnol. 2018 Jun 6;34(6):85. doi: 10.1007/s11274-018-2466-z.
For decades, diverse plasmid vectors have been continuously developed for molecular cloning of DNA fragment in the bacterial host cell Escherichia coli. Even with deliberate performances in vector preparation, the cloning approaches still face inevitable background colonies, or false positive clones, that may be arisen from intact or self-ligated plasmid molecules. To assist in such problem, two plasmids, pBS2ndd and pBS3ndd, which resistant to ampicillin and kanamycin respectively, were developed in this study as more advantageous cloning vector. The plasmids carry ndd, a lethal gene from bacteriophage T4 coding for nucleoid disruption protein that binds to the host chromosome and progressively kill the cell. The deadly toxicity of Ndd inhibits host cells that obtain intact or ndd-religated vector from growing, which results in low background and dramatically reduces the effort for selection of recombinants. Moreover, their identical multiple cloning site was designed to support various cloning strategies. Digestion of plasmids with XcmI allows for in vitro T/A ligation, while with EcoRV permits blunt-end ligation, with capability of blue-white colony screening. In vivo homologous recombination cloning is also utilizable by amplification of insert fragments using primers containing homology arms and transformation into capable E. coli strains. To demonstrate their advantages, the plasmids were used to clone PCR product samples for DNA sequencing with low-background and versatile cloning strategies. Such rapid and cost-effective cloning procedures are also proposed here. Finally, the cloning for protein expression with blue-white selection was also possible using egfp as a model regulated by lac and T7 promoters on the plasmid or other build-in promoters with the insert.
几十年来，人们不断开发各种质粒载体，用于在大肠杆菌等细菌宿主细胞中克隆 DNA 片段。即使在载体制备方面精心设计，克隆方法仍然面临不可避免的背景菌落或假阳性克隆，这些可能是完整或自我连接的质粒分子引起的。为了帮助解决这个问题，本研究开发了两种质粒，pBS2ndd 和 pBS3ndd，它们分别对氨苄青霉素和卡那霉素有抗性，作为更有利的克隆载体。这些质粒携带 ndd，这是来自噬菌体 T4 的一个致死基因，编码核破坏蛋白，与宿主染色体结合并逐渐杀死细胞。Ndd 的致命毒性抑制了从生长中获得完整或 ndd 连接载体的宿主细胞，从而降低了背景并大大减少了重组体选择的工作量。此外，它们相同的多克隆位点设计支持各种克隆策略。用 XcmI 消化质粒允许体外 T/A 连接，而用 EcoRV 允许平头末端连接，并具有蓝白菌落筛选能力。通过包含同源臂的引物扩增插入片段并转化到有能力的大肠杆菌菌株中，也可以进行体内同源重组克隆。为了证明它们的优势，我们使用这些质粒进行了 PCR 产物样本的克隆，用于 DNA 测序，采用了低背景和多种克隆策略。这里还提出了这种快速且具有成本效益的克隆程序。最后，还可以使用 egfp 作为模型，通过质粒上的 lac 和 T7 启动子或其他内置启动子进行蓝白选择的蛋白质表达克隆。
