Escherichia coli strains that allow antibiotic-free plasmid selection and maintenance by repressor titration
Escherichia coli strains that allow antibiotic-free plasmid selection and maintenance by repressor titration
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DOI：
10.1093/nar/29.5.e26
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发表时间：
2001-03-01
影响因子：
14.9
通讯作者：
Sherratt, David J.
中科院分区：
文献类型：
--
作者：
Cranenburgh, Rocky M.;Hanak, Julian A. J.;Sherratt, David J.
我们报告了两种新型大肠杆菌菌株（DH1lacdapD 和 DH1lacP2dapD）的构建，它们有助于在复杂培养基中无抗生素选择和稳定维持重组质粒。它们含有必需的染色体基因 dapD，受 lac 操纵子/启动子控制。除非补充 IPTG（诱导 dapD 表达）或 DAP，否则这些细胞会裂解。然而，当用含有 lac 操纵子的多拷贝质粒转化菌株时，操纵子竞争性滴定 LacI 阻遏物，并允许 lac 启动子表达 dapD。因此，可以通过抑制子滴定选择简单地通过其在任何培养基上生长的能力来分离和繁殖转化体。质粒上不需要抗生素抗性基因或其他蛋白质表达序列，并且质粒选择不需要抗生素，这使得这些菌株成为治疗性 DNA 和重组蛋白质生产的宝贵工具。我们描述了这些菌株的构建，并使用旨在促进重组 DNA 开发的新 pORT 质粒载体，通过阻遏物滴定演示了质粒选择和维持。
We report the construction of two novel Escherichia coli strains (DH1lacdapD and DH1lacP2dapD) that facilitate the antibiotic-free selection and stable maintenance of recombinant plasmids in complex media. They contain the essential chromosomal gene, dapD, under the control of the lac operator/promoter. Unless supplemented with IPTG (which induces expression of dapD) or DAP, these cells lyse. However, when the strains are transformed with a multicopy plasmid containing the lac operator, the operator competitively titrates the LacI repressor and allows expression of dapD from the lac promoter. Thus transformants can be isolated and propagated simply by their ability to grow on any medium by repressor titration selection. No antibiotic resistance genes or other protein expressing sequences are required on the plasmid, and antibiotics are not necessary for plasmid selection, making these strains a valuable tool for therapeutic DNA and recombinant protein production. We describe the construction of these strains and demonstrate plasmid selection and maintenance by repressor titration, using the new pORT plasmid vectors designed to facilitate recombinant DNA exploitation.