无痕敲除法构建食品安全级枯草芽孢杆菌无芽孢菌株

Construction of a food safe grade Bacillus subtilis nonspore strain with clean deletion

摘要: 利用同源重组法快速构建枯草芽孢杆菌(Bacilus subtilis168)spo0A基因缺陷型无芽孢菌株B.subtilis168Δspo0A。经PCR及核酸电泳验证,孢子形成率鉴定和芽孢染色镜检,确定最终获得不产芽孢的B.subtilis168Δspo0A基因缺失突变菌株。基因敲除完成后,菌株基因组不需引入抗性基因或其它任何筛选标记,实现了对spo0A基因的无痕基因敲除。本研究对枯草杆菌的工业化生产及提高相关产品食品质量安全性具有重要意义。

Abstract: Construction a nonspore B.subtilis 168 strain( B.subtilis 168Δspo0A) by deleting the spo0 A gene using the method of homologous recombination.The nonspore B.subtilis168Δspo0A strain was successfully got through PCR,nucleic acid electrophoresis,sporulation ratio and microscopy identification.The genome of this engineering strain does not need to insert resistance gene or any other selection marker gene after spo0 A genes' knockout which called clean deletion. This study is of great significance for industrial production of B.subtilis as well as improve safety of food and drug.