利用框内缺失法构建变异链球菌srtA基因缺失株

ObjectiveTo construct srtA-gene deletion mutant ofStreptococcus mutans(S. mutans) UA159 with IFDC2 cassette through overlapping polymerase chain reaction (PCR) and allelic homologous recombination.MethodsFirst, the upstream and downstream fragments surrounding the srtA and IFDC2 cassette were PCR amplified and ligated through over-lapping 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.ResultsThe 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.ConclusionThe markerless srtA-deletion mutant ofS. mutanswas constructed successfully, which laid a foundation for further study of its biological function and influence on the biofilm formation ofS. mutans.