Construction of P.gingivalis mutans strains
Porphyromonas gingivalis genomece data were obtained from The Kyoto Encyclopedia of Genes and Genomes web site to construct mutans.
The P.gingivalis rgpA mutans (RK103) was constructed as follows: a DNA fragment corresponding to a 1.4-kb region rgpA was generated by PCR
using with a forward primer ,5'-GGGGGGAATTCGGAGAGATCGCCACGCTTGATGATCCTTTT, containing an EcoRI site (underlined)and a reverse primer,5'-GGGGGGGATCCTCAGACCTGTCAGATTGATTGTAGCTGTTC,contraining a BamHI site (underlined).
The resulting fragment was cloned into the pCR4
vector (Invitrogen, Carlsbad, CA) and the plasmid was designated as pRK1. An ermF-ermAM DNA fragment (Fletcher etal., 1995) was inserted into an EcoRV site of the
rgpA fragment in pRK1,resulting in pRK2. pRK2 plasmid DNA was then linearized by Notl digestion and introduced into the P.gingivalis kgp mutant (KDP129) cells
by electroporation (Kikuchi et al.,2005)
The transformants were spread on Tryptic soy agar plates containing 10â╩gmL-1 Em, 20â╩gmL-1 Cm, 5â╩gmL-1 hemin and 0.5â╩gmL-1menadione,and incubated anaerobically for
7 days. Insertion of the Em-resistant cassette following transformation(RK103) was verified by Southern and Western blot analyses.