Wolf Katerina, Rahnama Mostafa, Fields Kenneth A
Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky College of Medicine, Lexington, KY, USA.
Methods Mol Biol. 2019;2042:151-164. doi: 10.1007/978-1-4939-9694-0_11.
Progress in understanding molecular mechanisms contributing to chlamydial pathogenesis has been greatly facilitated by recent advances in genetic manipulation of C. trachomatis. Valuable approaches such as random, chemically induced mutagenesis or targeted, insertion-based gene disruption have led to significant discoveries. We describe herein a technique for generating definitive null strains via complete deletion of chromosomal genes in C. trachomatis. Fluorescence-reported allelic exchange mutagenesis (FRAEM), using the suicide vector pSUmC, enables targeted deletion of desired chromosomal DNA. The protocol provided here describes steps required to produce transformation competent chlamydiae, generate a specific allelic exchange plasmid construct, carry out mutagenesis, and isolate clonal populations of resulting mutant strains.
沙眼衣原体基因操作的最新进展极大地推动了对衣原体致病分子机制理解的进程。诸如随机化学诱变或基于插入的靶向基因破坏等有价值的方法已带来了重大发现。我们在此描述一种通过完全删除沙眼衣原体染色体基因来产生确定的无功能菌株的技术。使用自杀载体pSUmC的荧光报告等位基因交换诱变（FRAEM）能够靶向删除所需的染色体DNA。此处提供的方案描述了产生具有转化能力的衣原体、构建特定的等位基因交换质粒、进行诱变以及分离所得突变菌株的克隆群体所需的步骤。
