Conditionally replicative and conjugative plasmids carrying lacZ alpha for cloning, mutagenesis, and allele replacement in bacteria
Conditionally replicative and conjugative plasmids carrying lacZ alpha for cloning, mutagenesis, and allele replacement in bacteria
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DOI：
10.1006/plas.1996.0001
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发表时间：
1996-01-01
期刊：
影响因子：
2.6
通讯作者：
Wanner, BL
中科院分区：
文献类型：
--
作者：
Metcalf, WW;Jiang, WH;Wanner, BL
我们描述了几种用于诱变和等位基因替换实验的新克隆载体。这些质粒具有 R6K gamma DNA 复制起点 (oriR(R6K gamma))，因此它们仅在提供 Pi 复制蛋白（由 pir 编码）的细菌中复制，并且可以通过使用编码野生型或突变型 Pi 的大肠杆菌菌株将它们维持在低或高质粒拷贝数。它们还携带 RP4 转移起点 (oriT(RP4))，因此可以通过与多种细菌接合来转移。它们中的大多数编码 lacZ α，用于对带有携带插入片段的质粒的集落进行蓝白色筛选，以及用于制备单链 DNA 的 fl DNA 复制起点。特定的质粒对于等位基因替换实验特别有用，因为它们还编码阳性反选择标记。一组携带 tetAR（来自 Tn10），可以将无质粒分离子作为四环素敏感 (Tet(S)) 重组体进行正选。另一组携带sacB（来自枯草芽孢杆菌），允许选择无质粒分离子作为蔗糖抗性（Suc(R)）分离子。因此，可以将这些质粒的衍生物引入非pir宿主中（通过接合转移、转化或电穿孔），并使用质粒抗生素抗性标记来选择通过同源序列重组到染色体中的质粒的整合体。随后可以选择具有等位基因替换的无质粒分离体作为Tet(S)或Suc(R)重组体。许多附加特征（包括侧翼有 T3 和 T7 RNA 聚合酶启动子的多个克隆位点的存在）使这些质粒也可用作通用克隆载体。 (C) 1996 学术出版社
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 fl 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 (Tet(S)) recombinants. Another set carries sacB (from Bacillus subtilis) that allows selecting plasmid-free segregants as sucrose-resistant (Suc(R)) 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 Tet(S) or Suc(R) 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. (C) 1996 Academic Press, Inc.