文献类型：专利

英文题名：Method for unmarked gene knockout of Pediococcus acidilactici DQ2 based on homologous recombination used as starting strain for gene knockout and transformation involves constructing plasmid and performing gene knockout and electroporation

作者：BAO J;ZHANG P;ZHANG J;TU Y;GAO Q

机构：[1]UNIV EAST CHINA SCI & TECHNOLOGY

申请号：CN105821071-A

申请日：2015-01-06

公开日：2016-08-03

语种：英文

收录：DERWENT

摘要：NOVELTY - Unmarked gene knockout of Pediococcus acidilactici DQ2 based on homologous recombination comprises constructing pSET4E plasmid by substituting erythromycin resistance gene with spectinomycin; performing gene knockout on target upstream and downstream homologous fragments, and cloning to pSET4E plasmid to obtain knockout plasmid of target gene; transforming into Pediococcus acidilactici DQ2 by electroporation to obtain knockout plasmid-containing strain; culturing to obtain first homologous recombination containing erythromycin; and culturing in absence of erythromycin. USE - Method for unmarked gene knockout of Pediococcus acidilactici DQ2 based on homologous recombination used as starting strain for subsequent gene knockout and transformation. ADVANTAGE - The method is safe and suitable large-scale industrial production. DETAILED DESCRIPTION - Unmarked gene knockout of Pediococcus acidilactici DQ2 based on homologous recombination comprises constructing pSET4E plasmid by substituting erythromycin resistance gene with spectinomycin, where the pSET4S plasmid is temperature-sensitive Escherichia coli gram-positive bacterium containing spectinomycin resistance genes; performing gene knockout on target upstream and downstream homologous fragments, and cloning to pSET4E plasmid to obtain knockout plasmid of target gene; transforming knockout plasmid into Pediococcus acidilactici DQ2 by electroporation to obtain knockout plasmid-containing strain; culturing at 42 degrees C while hindering plasmid replication to obtain first homologous recombination containing erythromycin; and culturing in absence of erythromycin at 28 degrees C, and continuing culture for 1-10 times under same conditions to obtain second homologous recombination without erythromycin.