The Deputy Lactobacillus Casei Hd1.7prck Gene Knockout Vector And Its Power Conversion Conditions Initially Established

A series of primers were designed and synthesized according to the relative fragments and the conservative domain of complete sequences of prcK and prcR of Lactobacillus paracasei HD1.7, which have been recorded by the GenBank. And the fragment of Histidine protein kinase, named prcK-550, was obtained from Lactobacillus paracasei HD1.7 by PCR and was cloned into pUC18 vector by transformant. In this study, to identificate the function of prcK gene, gene knock-out vector named pUC18-prcK-550-tet were constructed by inserting a tetracycline-resistant gene into prcK-550, whose single site was breakaged by enzymatic digestion of ClaI .Then the vector was transformed into E.coli DH5α, and the transformed bacteria was resistant to tetracycline.The optimized parameters affecting high-efficent electrotransforming of plasmid pMG36e into Lactobacillus paracasei HD1.7 were studied preliminarily. It showed that a highest electro-transformation efficiency could be obtained when the recipient cell was treated by 1% of glycine and washed by electrotransformation buffer of AEB1, as well as the 9 kV/cm of field strength and one time of pulse with the OD600nm value of 0.78.The purpose of this study is to construct the prcK knock-out vector by cloning gene of Histidine protein kinase(prcK), which was one of the putative quorum sensing genes. And the preliminary study on the electrotransformation condition of HD 1.7 laid the foundation for the construction of prcK gene deletion mutation and reversal mutation and the further studies on the function of prcK.