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Oral Microbiol Immunol. 2006 Apr;21(2):132-5.

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

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Department of Oral Biology, College of Dentistry, University of Illinois at Chicago, Chicago, IL 60612, USA.


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%.

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