Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange.
Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange.
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DOI：
10.1038/nprot.2015.115
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发表时间：
2015-11
期刊：
影响因子：
14.8
通讯作者：
Harrison JJ
中科院分区：
文献类型：
--
作者：
Hmelo LR;Borlee BR;Almblad H;Love ME;Randall TE;Tseng BS;Lin C;Irie Y;Storek KM;Yang JJ;Siehnel RJ;Howell PL;Singh PK;Tolker-Nielsen T;Parsek MR;Schweizer HP;Harrison JJ
等位基因交换是细菌基因组工程的一种有效方法。该方案描述了使用该技术在铜绿假单胞菌中进行基因敲除和敲入，以及单核苷酸插入、缺失和取代。与等位基因交换的其他方法不同，该方案不需要异源重组酶来插入或切除来自靶染色体的选择性标记。相反，正选择和负选择仅通过自杀载体编码的功能和宿主细胞蛋白实现。在这里，突变等位基因，其侧翼与受体染色体同源的区域，在体外合成，然后使用标准程序克隆到等位基因交换载体中。然后通过接合将这些自杀载体引入受体细胞。同源重组然后导致抗生素抗性单交换突变体，其中质粒已经位点特异性地整合到染色体中。随后，使用蔗糖介导的反选择直接分离未标记的双交换突变体。这个两步过程产生精确到DNA的单个碱基对的无缝突变。整个过程需要约2周。
Allelic exchange is an efficient method of bacterial genome engineering. This protocol describes the use of this technique to make gene knockouts and knockins, as well as single nucleotide insertions, deletions and substitutions in Pseudomonas aeruginosa. Unlike other approaches to allelic exchange, this protocol does not require heterologous recombinases to insert or excise selective markers from the target chromosome. Rather, positive and negative selection are enabled solely by suicide vector-encoded functions and host cell proteins. Here, mutant alleles, which are flanked by regions of homology to the recipient chromosome, are synthesized in vitro and then cloned into allelic exchange vectors using standard procedures. These suicide vectors are then introduced into recipient cells by conjugation. Homologous recombination then results in antibiotic resistant single-crossover mutants in which the plasmid has integrated site-specifically into the chromosome. Subsequently, unmarked double-crossover mutants are isolated directly using sucrose-mediated counter-selection. This two-step process yields seamless mutations that are precise to a single base pair of DNA. The entire procedure requires ~2 weeks.