[Construction of srtA-deletion mutant of Streptococcus mutans by an in-frame deletion system]

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.