Metcalf W W, Jiang W, Daniels L L, Kim S K, Haldimann A, Wanner B L
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907, USA.
Plasmid. 1996 Jan;35(1):1-13. doi: 10.1006/plas.1996.0001.
We describe several new cloning vectors for mutagenesis and allele replacement experiments. These plasmids have the R6K gamma DNA replication origin (oriR(R6K gamma) so they replicate only in bacteria supplying the pi replication protein (encoded by pir), and they can be maintained at low or high plasmid copy number by using Escherichia coli strains encoding either wild-type or mutant forms of pi. They also carry the RP4 transfer origin (oriT(RP4)) so they can be transferred by conjugation to a broad range of bacteria. Most of them encode lacZ alpha for blue-white color screening of colonies for ones with plasmids carrying inserts, as well as the f1 DNA replication origin for preparation of single-stranded DNA. Particular plasmids are especially useful for allele replacement experiments because they also encode a positive counterselectable marker. One set carries tetAR (from Tn10) that allows for positive selection of plasmid-free segregants as tetracycline-sensitive (TetS) recombinants. Another set carries sacB (from Bacillus subtilis) that allows selecting plasmid-free segregants as sucrose-resistant (SucR) ones. Accordingly, derivatives of these plasmids can be introduced into a non-pir host (via conjugative transfer, transformation, or electroporation), and integrants with the plasmid recombined into the chromosome via homologous sequences are selected using a plasmid antibiotic resistance marker. Plasmid-free segregants with an allele replacement can be subsequently selected as TetS or SucR recombinants. A number of additional features (including the presence of multiple cloning sites flanked by T3 and T7 RNA polymerase promoters) make these plasmids useful as general cloning vectors as well.
我们描述了几种用于诱变和等位基因替换实验的新型克隆载体。这些质粒具有R6Kγ DNA复制起点（oriR(R6Kγ)），因此它们仅在提供π复制蛋白（由pir编码）的细菌中复制，并且通过使用编码野生型或突变形式π的大肠杆菌菌株，它们可以以低或高质粒拷贝数维持。它们还携带RP4转移起点（oriT(RP4)），因此可以通过接合转移到多种细菌中。它们中的大多数编码lacZα用于对携带插入片段的质粒菌落进行蓝白斑筛选，以及用于制备单链DNA的f1 DNA复制起点。特定的质粒对于等位基因替换实验特别有用，因为它们还编码一个正向反选择标记。一组携带tetAR（来自Tn10），可将无质粒的分离株作为四环素敏感（TetS）重组体进行正向选择。另一组携带sacB（来自枯草芽孢杆菌），可将无质粒的分离株作为蔗糖抗性（SucR）分离株进行选择。因此，这些质粒的衍生物可以引入非pir宿主（通过接合转移、转化或电穿孔），并使用质粒抗生素抗性标记选择通过同源序列与染色体重组的质粒整合体。随后可以将具有等位基因替换的无质粒分离株选择为TetS或SucR重组体。许多其他特征（包括存在由T3和T7 RNA聚合酶启动子侧翼的多个克隆位点）也使这些质粒可用作通用克隆载体。
