沙门菌基因突变株的构建方法研究
Optimization of the method for construction of Salmonella spp. Mutant
叶颖 1邱桥成 2李嫄渊 1吴淑燕 1黄瑞1
作者信息
- 1. 苏州大学医学部病原生物学系,江苏苏州215123
- 2. 苏州大学医学部病原生物学系,江苏苏州215123;苏州大学附属第一医院、江苏省血液研究所,江苏苏州215006
- 折叠
摘要
目的 研究沙门菌基因突变株构建的方法,为利用基因工程技术对细菌进行遗传性状改造,深入研究沙门菌防治手段提供工具.方法 选用不同感受态细菌培养时间、培养基、感受态细胞浓度和电击参数等条件,以小分子质粒pMDT-GFP电转化受试菌,通过计算转化效率确定优化的电转条件.在此基础上用大分子质粒pGBM151-spvB电转化受试菌,再以蔗糖诱导同源重组,筛选突变株.结果 以含15 g/LNaCl的高渗LB培养基培养细菌至A600为0.3,制备浓度为1010cfu/ml的感受态细胞,在参数为2.5 kV、400 Ω、50 μF条件下,小质粒pMDT-GFP电转鼠伤寒沙门菌可获得高转化效率.以往难以转化的大质粒pGBM151-spvB在该条件下成功获得转化子,进而成功构建突变株.结论 上述方法能用于高效构建沙门菌基因突变株.
Abstract
Objective To investigate the method of Salmonella spp. mutant construction, optimizes the processing and provides the basis for further experiments in genetically modification. Methods Two different plasmid vectors DNAs pMDT-GFP and pGBM151-spvB were electrotransformed into S. typhimuri-um under various conditions including growth stage, medium, concentration of competent cells and pulse strength. The transformation efficiency changes were discovered and the optimized conditions were evaluated accordingly. Results The optimal electrotransformation effect can be obtained when A600 is 0. 3 , the medium is hypertonic LB contained 15g/L NaCl, concentration of competent cells is 1010 cfu/ml and the electroporation parameters is 2.5 kV, 400 Ω,50 μF. Conclusion This method can be used to efficiently of construct Salmonella spp. mutant.
关键词
沙门菌/基因突变/转化Key words
Salmonella spp./gene mutation/transformation引用本文复制引用
基金项目
国家自然科学基金(30972768)
江苏省自然科学基金(L2134051511)
江苏省"青蓝工程"项目(SR13400211)
出版年
2012
NETL
NSTL
维普
万方数据