Xuan Chen, Haixia Liu, Xian Peng, Ling Zou
State Key Laboratory of Oral Diseases & Dept. of Conservative Dentistry and Endodontics, National Clinical Research Center for Oral Diseases & West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China.
Hua Xi Kou Qiang Yi Xue Za Zhi. 2017 Dec 1;35(6):588-592. doi: 10.7518/hxkq.2017.06.005.
To construct srtA-gene deletion mutant of Streptococcus mutans (S. mutans) UA159 with IFDC2 cassette through overlapping polymerase chain reaction (PCR) and allelic homologous recombination.
First, the upstream and downstream fragments surrounding the srtA and IFDC2 cassette were PCR amplified and ligated through overlapping PCR. The resulting amplicon was transformed into UA159, and positive transformants were selected on BHI plates containing erythromycin. Second, upstream and downstream fragments of srtA with overlap regions were generated by PCR and were overlapped to create upΔ-down amplicon. Then, the upΔ-down amplicon was transformed into the aforementioned positive transformants and selected on BHI plates containing p-Cl-Phe.
The PCR analysis and DNA sequencing results indicated that the coding region of the srtA was completely deleted, and the upstream and downstream regions flanking the srtA were ligated seamlessly.
The markerless srtA-deletion mutant of S. mutans was constructed successfully, which laid a foundation for further study of its biological function and influence on the biofilm formation of S. mutans.
通过重叠聚合酶链反应（PCR）和等位基因同源重组构建携带IFDC2盒的变形链球菌（S. mutans）UA159的srtA基因缺失突变体。
首先，对srtA和IFDC2盒周围的上游和下游片段进行PCR扩增，并通过重叠PCR进行连接。将所得扩增产物转化到UA159中，并在含有红霉素的BHI平板上筛选阳性转化体。其次，通过PCR产生具有重叠区域的srtA的上游和下游片段，并使其重叠以产生upΔ-down扩增产物。然后，将upΔ-down扩增产物转化到上述阳性转化体中，并在含有对氯苯丙氨酸的BHI平板上进行筛选。
PCR分析和DNA测序结果表明，srtA的编码区被完全删除，且srtA两侧的上游和下游区域无缝连接。
成功构建了无标记的变形链球菌srtA缺失突变体，为进一步研究其生物学功能及其对变形链球菌生物膜形成的影响奠定了基础。
