Unmarked gene deletion mutagenesis of gtfB and gtfC in Streptococcus mutans using a targeted hit-and-run strategy with a thermosensitive plasmid

Atlagic D., Kilic A. O. , Tao L.

ORAL MICROBIOLOGY AND IMMUNOLOGY, vol.21, no.2, pp.132-135, 2006 (Peer-Reviewed Journal) identifier identifier identifier

  • Publication Type: Article / Article
  • Volume: 21 Issue: 2
  • Publication Date: 2006
  • Doi Number: 10.1111/j.1399-302x.2006.00267.x
  • Journal Indexes: Science Citation Index Expanded, Scopus
  • Page Numbers: pp.132-135


Unmarked gene deletion of the Streptococcus mutans gtfB-gtfC locus was achieved using a thermosensitive plasmid. DNA fragments flanking the locus were amplified by polymerase chain reaction and jointly ligated into pG+host5, which was transformed into S. mutans at 37 degrees C to facilitate integration. A transformant was then grown at 28 degrees C for 60 generations without antibiotics to facilitate excision. Antibiotic sensitive clones appeared at a frequency of about 99% and were analyzed for deletions of gtfB, gtfC and a part of mbrA by the lack of insoluble glucan synthesis, sensitivity to bacitracin, and polymerase chain reaction. Targeted gene deletions occurred at a frequency of 2.5%.