抄録
Genetic manipulation techniques for Bifidobacterial strains, such as transformation and gene-knockout, have been considered ‘tough nuts to crack’. We have developed new tools to make breakthroughs in this regard. The generally low transformation efficiency has been overcome by a new concept of ‘plasmid artificial modification’, which protects external plasmids against host restriction systems. We also constructed a temperature sensitive plasmid, pKO403, which can be cured at 42 °C. Positive/negative bi-functional selection has been developed with a focus on the pyrE gene, encoding orotate phosphoribosyl transferase, which can be positively selected on uracil(-) medium and negatively selected with 5-fluoroorotic acid. Combining these newly developed tools, marker-less gene-knockout can be performed at the practical routine level. Multi-gene knockout is also feasible by repetition of these techniques.
