Zhao Xingxing, Cheng Yumei, Wu Changxue, Ren Wei, Rao Fengqin, Zhou Qian, Cui Guzhen, Qi Xiaolan, Hong Wei
Key Laboratory of Endemic and Ethnic Diseases, Ministry of Education, Guizhou Medical University, Guiyang 550004, Guizhou, China.
Department of ICU, The Affiliate Hospital of Guizhou Medical University, Guiyang 550004, Guizhou, China.
Sheng Wu Gong Cheng Xue Bao. 2020 Aug 25;36(8):1659-1671. doi: 10.13345/j.cjb.190547.
To construct TeI3c/4c-based and temperature-inducible gene inactivation system (Thermotargetron) and to apply it to gene inactivation of mesophilic bacteria. The subunit of flagellum (fliC) and C4 dicarboxylate orotate:H⁺ symporter (dctA) genes were chosen as targets in the genome of Escherichia coli HMS174 (DE3) strain. According to recognition roles of TeI3c/4c intron, the fliC489a, fliC828s, fliC1038s and dctA2a sites were chosen as target sites. Gene-targeting plasmids were constructed based on pHK-TT1A by using overlap PCR method and transformed into HMS174 cells. An aliquot mid-log phase cultures of the transformants were shocked at 48 °C and plated on LB plate (containing chloramphenicol). Afterwards, gene mutants were screened by using colony PCR and DNA sequencing. After the mutants were obtained, the phenotypes of ΔfliC and ΔdctA gene mutants were characterized by using agar puncture and carbon metabolism experiments. Colony PCR and sequencing results show that TeI3c/4c intron was inserted in the designed sites of fliC and dctA genes. The gene-targeting efficiency of Thermotargetron system was 100%. Phenotype verification experiments of the mutants demonstrated that the cell motility of all ΔfliC mutants was damaged and the malate assimilation ability of ΔdctA mutant was deprived comparing to wild-type HMS174 strain. In our study, a temperature-inducible and high-efficiency gene inactivation system was established for mesophilic bacteria. This system could achieve high efficiency and precise gene inactivation by modulation of the incubation duration of the transformants at 48 °C.
构建基于TeI3c/4c的温度诱导基因失活系统（热靶向子）并将其应用于嗜温菌的基因失活。选择鞭毛亚基（fliC）和C4二羧酸乳清酸：H⁺同向转运体（dctA）基因作为大肠杆菌HMS174（DE3）菌株基因组中的靶标。根据TeI3c/4c内含子的识别作用，选择fliC489a、fliC828s、fliC1038s和dctA2a位点作为靶位点。利用重叠PCR方法基于pHK-TT1A构建基因靶向质粒，并将其转化到HMS174细胞中。将转化子的等分对数中期培养物在48℃下进行热激处理，然后接种到含有氯霉素的LB平板上。之后，通过菌落PCR和DNA测序筛选基因突变体。获得突变体后，利用琼脂穿刺和碳代谢实验对ΔfliC和ΔdctA基因突变体的表型进行表征。菌落PCR和测序结果表明，TeI3c/4c内含子插入到了fliC和dctA基因的设计位点。热靶向子系统的基因靶向效率为100%。突变体表型验证实验表明，与野生型HMS174菌株相比，所有ΔfliC突变体的细胞运动能力均受损，ΔdctA突变体的苹果酸同化能力丧失。在本研究中，为嗜温菌建立了一种温度诱导高效基因失活系统。该系统可通过调节转化子在48℃下的培养时间实现高效、精确的基因失活。
