PDF(Pubmed)
Abstract：
Over the past years, several methods have been developed for gene cloning. Choosing a cloning strategy depends on various factors, among which simplicity and affordability have always been considered. The aim of this study, on the one hand, is to simplify gene cloning by skipping in vitro assembly reactions and, on the other hand, to reduce costs by eliminating relatively expensive materials. We investigated a cloning system using Escherichia coli harboring two plasmids, pLP-AmpR and pScissors-CmR. The pLP-AmpR contains a landing pad (LP) consisting of two genes (λ int and λ gam) that allow the replacement of the transformed linear DNA using site-specific recombination. After the replacement process, the inducible expressing SpCas9 and specific sgRNA from the pScissors-CmR (CRISPR/Cas9) vector leads to the removal of non-recombinant pLP-AmpR plasmids. The function of LP was explored by directly transforming PCR products. The pScissors-CmR plasmid was evaluated for curing three vectors, including the origins of pBR322, p15A, and pSC101. Replacing LP with a PCR product and fast-eradicating pSC101 origin-containing vectors was successful. Recombinant colonies were confirmed following gene replacement and plasmid curing processes. The results made us optimistic that this strategy may potentially be a simple and inexpensive cloning method. KEY POINTS: •The in vivo cloning was performed by replacing the target gene with the landing pad. •Fast eradication of non-recombinant plasmids was possible by adapting key vectors. •This strategy is not dependent on in vitro assembly reactions and expensive materials.
摘要：
在过去的几年里,已经开发了几种基因克隆方法。选择克隆策略取决于各种因素，其中，简单性和可负担性一直被考虑。本研究的目的,一方面,是通过跳过体外组装反应来简化基因克隆，另一方面,通过消除相对昂贵的材料来降低成本。我们研究了一个克隆系统，使用带有两个质粒的大肠杆菌，pLP-AmpR和pScissors-CmR。pLP-AmpR包含由两个基因（λint和λgam）组成的着陆区（LP），该基因允许使用位点特异性重组替换转化的线性DNA。在更换过程之后,例如,来自pScissors-CmR(CRISPR/Cas9)载体的可诱导表达SpCas9和特异性sgRNA导致非重组pLP-AmpR质粒的去除。通过直接转化PCR产物来探索LP的功能。评价pScissors-CmR质粒对三种载体的固化作用,包括pBR322，p15A，pSC101用PCR产物替换LP并快速根除含有pSC101来源的载体是成功的。在基因置换和质粒固化过程后确认重组菌落。结果使我们乐观地认为，这种策略可能是一种简单而廉价的克隆方法。关键点:•通过用着陆垫替换靶基因进行体内克隆。•通过适应关键载体,非重组质粒的快速根除是可能的。•该策略不依赖于体外组装反应和昂贵的材料。
