Chen Lei, Ge Xiuchun, Xu Ping
Philips Institute, Virginia Commonwealth University, Richmond, VA, USA.
Methods Mol Biol. 2015;1279:15-23. doi: 10.1007/978-1-4939-2398-4_2.
Essential genes in pathogens are important for the development of antibacterial drugs. In this report, we described a protocol to identify essential genes in the Streptococcus sanguinis SK36 strain using genome-wide deletion mutation. A fusion PCR-based method is used to construct gene deletion fragments, which contain kanamycin resistance cassettes with two flanking arms of DNA upstream and downstream of the target gene. The linear fused PCR amplicons were transformed into S. sanguinis SK36 cells. No kanamycin-resistant transformants suggested the gene essentiality because the deletion of the essential gene renders a lethal phenotype of the transformants. The putative essential genes were further confirmed by independent transformations up to five attempts. The false nonessential genes were also identified by removing double-band mutants.
病原体中的必需基因对抗菌药物的研发至关重要。在本报告中，我们描述了一种使用全基因组缺失突变来鉴定血链球菌SK36菌株中必需基因的方案。基于融合PCR的方法用于构建基因缺失片段，其包含卡那霉素抗性盒以及靶基因上下游的两个侧翼DNA臂。线性融合PCR扩增产物被转化到血链球菌SK36细胞中。没有卡那霉素抗性转化子表明该基因是必需的，因为必需基因的缺失会导致转化子出现致死表型。通过多达五次独立转化进一步确认推定的必需基因。还通过去除双带突变体鉴定了假非必需基因。
