Caguiat Jonathan James
Department of Biological Sciences, Center for Applied Chemical Biology, Youngstown State University;
J Vis Exp. 2014 Oct 31(92):e51934. doi: 10.3791/51934.
Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can be generated using a transposome. The commercially available, Tn5-derived transposome used in this protocol consists of a linear segment of DNA containing an R6Kγ replication origin, a gene for kanamycin resistance and two mosaic sequence ends, which serve as transposase binding sites. The transposome, provided as a DNA/transposase protein complex, is introduced by electroporation into the prototrophic strain, Enterobacter sp. YSU, and randomly incorporates itself into this host's genome. Transformants are replica plated onto Luria-Bertani agar plates containing kanamycin, (LB-kan) and onto M-9 medium agar plates containing kanamycin (M-9-kan). The transformants that grow on LB-kan plates but not on M-9-kan plates are considered to be auxotrophs. Purified genomic DNA from an auxotroph is partially digested, ligated and transformed into a pir+ Escherichia coli (E. coli) strain. The R6Kγ replication origin allows the plasmid to replicate in pir+ E. coli strains, and the kanamycin resistance marker allows for plasmid selection. Each transformant possesses a new plasmid containing the transposon flanked by the interrupted chromosomal region. Sanger sequencing and the Basic Local Alignment Search Tool (BLAST) suggest a putative identity of the interrupted gene. There are three advantages to using this transposome mutagenesis strategy. First, it does not rely on the expression of a transposase gene by the host. Second, the transposome is introduced into the target host by electroporation, rather than by conjugation or by transduction and therefore is more efficient. Third, the R6Kγ replication origin makes it easy to identify the mutated gene which is partially recovered in a recombinant plasmid. This technique can be used to investigate the genes involved in other characteristics of Enterobacter sp. YSU or of a wider variety of bacterial strains.
原养型细菌能在添加了葡萄糖的M-9基本盐培养基（M-9培养基）上生长，葡萄糖作为碳源和能源。营养缺陷型菌株可以通过转座体产生。本实验方案中使用的市售Tn5衍生转座体由一段线性DNA片段组成，该片段包含一个R6Kγ复制起点、一个卡那霉素抗性基因和两个镶嵌序列末端，这两个末端作为转座酶结合位点。作为DNA/转座酶蛋白复合物提供的转座体通过电穿孔导入原养型菌株肠道杆菌YSU，并随机整合到该宿主的基因组中。将转化子影印接种到含有卡那霉素的Luria-Bertani琼脂平板（LB-卡那平板）和含有卡那霉素的M-9培养基琼脂平板（M-9-卡那平板）上。在LB-卡那平板上生长但在M-9-卡那平板上不生长的转化子被认为是营养缺陷型菌株。从营养缺陷型菌株中纯化的基因组DNA进行部分酶切、连接，然后转化到pir + 大肠杆菌菌株中。R6Kγ复制起点使质粒能够在pir + 大肠杆菌菌株中复制，卡那霉素抗性标记用于质粒筛选。每个转化子都拥有一个新的质粒，该质粒包含侧翼为中断染色体区域的转座子。桑格测序和基本局部比对搜索工具（BLAST）可推测出中断基因的假定身份。使用这种转座体诱变策略有三个优点。第一，它不依赖宿主中转座酶基因的表达。第二，转座体通过电穿孔导入目标宿主，而不是通过接合或转导，因此效率更高。第三，R6Kγ复制起点便于识别在重组质粒中部分恢复的突变基因。该技术可用于研究参与肠道杆菌YSU或更广泛细菌菌株其他特性的基因。
