Construction Of The Gene Knock-out System For Propionibacterium Freudenreichii

We isolated four Propionibacteriums from the cheese with anaerobic technology in this experiment, then we extracted the DNA from Propionibacterium with liquid nitrogen grinding method. Determing two separation of strains are Propionibacterium freudenreichii subsp.shermanii and the other two separation of strains are Propionibacterium freudenreichii subsp. freudenreichii, we amply the conserved region of16S rDNA using the universal primers1492r and27f. This experiment we construct the gene knockout system of Propionibacterium with Propionibacterium freudenreichii subsp.freudenreichii.The separation of strain as the experimental strain, our experiment amplifies the upstream and downstream fragments of hemE by PCR, and we construct a target plasmid including homological arms and hygB resistance gene.The targeting plasmid introduct the Propionibacterium competent cells and subcultured it for generations. Finally, the homologous recombination verified between targeting plasmid and Propionibacterium acnes by PCR. This study will give the new skills of Propionibacterium and lay the foundation of methodology of improving its entire metabolic pathway.