[Construction PG0839 gene-defective mutant of Porphyromonas gingivalis].

Abstract

In order to determine the function of PG0839 gene from Porphyromonas gingivalis (P. gingivalis) W83 strains, we intended to create a mutant in the PG0839 gene by homologous recombination.
1 584bp PG0839 gene fragment was amplified, digested by BamH I and EcoR I, purified and ligated to pUC19. The recombinant plasmid was designated as pPG0839-1. The erm cassette (2 101 bp) was inserted into the EcoR V restriction site of the PG0839 gene. The resultant recombinant plasmid, pPG0839-2, was used as a donor in the electroporation of P. gingivalis W83. After electroporated and selected on erythromycin brain heart infusion plates, a single colony was collected and designated as PG0839 gene-defective mutant.
A mutant in PG0839 gene was created by insertional inactivation, and inactivation of PG0839 gene was confirmed by restriction endonuclease digestive, sequencing, polymerase chain reaction (PCR) and reverse transcription PCR.
A PG0839 gene-defective mutant was created successfully.