Gene doctoring is an efficient recombination-based genetic engineering approach to mutagenesis of the bacterial chromosome that combines the λ-Red recombination system with a suicide donor plasmid that is cleaved in vivo to generate linear DNA fragments suitable for recombination. The use of a suicide donor plasmid makes Gene Doctoring more efficient than other recombineering technologies. However, generation of donor plasmids typically requires multiple cloning and screening steps. We constructed a simplified acceptor plasmid, called pDOC-GG, for the assembly of multiple DNA fragments precisely and simultaneously to form a donor plasmid using Golden Gate assembly. Successful constructs can easily be identified through blue-white screening. We demonstrated proof of principle by inserting a gene for green fluorescent protein into the chromosome of Escherichia coli. We also provided related genetic parts to assist in the construction of mutagenesis cassettes with a tetracycline-selectable marker. Our plasmid greatly simplifies the construction of Gene Doctoring donor plasmids and allows for the assembly of complex, multi-part insertion or deletion cassettes with a free choice of target sites and selection markers. The tools we developed are applicable to gene editing for a wide variety of purposes in Enterobacteriaceae and potentially in other diverse bacterial families.

中文翻译：

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用于基因修复的金门构建体的创建

基因修复是一种有效的基于重组的基因工程方法来诱变细菌染色体，它将 λ-Red 重组系统与在体内切割的自杀供体质粒相结合，以生成适合重组的线性 DNA 片段。使用自杀供体质粒使基因修复比其他重组技术更有效。然而，供体质粒的产生通常需要多个克隆和筛选步骤。我们构建了一个简化的受体质粒，称为 pDOC-GG，用于精确并同时组装多个 DNA 片段，以使用金门组装形成供体质粒。通过蓝白筛选可以轻松识别成功的构建体。我们通过将绿色荧光蛋白基因插入大肠杆菌的染色体来证明原理。我们还提供了相关的遗传部分，以帮助构建具有四环素选择性标记的诱变盒。我们的质粒极大地简化了 Gene Doctoring 供体质粒的构建，并允许组装复杂的多部分插入或删除盒，并可以自由选择靶位点和选择标记。我们开发的工具适用于肠杆菌科和其他不同细菌科的基因编辑，用途广泛。我们的质粒极大地简化了 Gene Doctoring 供体质粒的构建，并允许组装复杂的多部分插入或删除盒，并可以自由选择靶位点和选择标记。我们开发的工具适用于肠杆菌科和其他不同细菌科的基因编辑，用途广泛。我们的质粒极大地简化了 Gene Doctoring 供体质粒的构建，并允许组装复杂的多部分插入或删除盒，并可以自由选择靶位点和选择标记。我们开发的工具适用于肠杆菌科和其他不同细菌科的基因编辑，用途广泛。