Departamento de Genética, Facultad de Biología, Universidad de Sevilla, 41080 Sevilla, Spain.
Université Paris-Saclay, CEA, CNRS, Institut de Biologie Intégrative de la Cellule (I2BC), 91190 Gif-sur-Yvette, France.
Cold Spring Harb Protoc. 2023 Sep 1;2023(9):651-662. doi: 10.1101/pdb.prot107950.
We describe a simple recombineering-based procedure for generating single-copy gene fusions to superfolder GFP (sfGFP) and monomeric Cherry (mCherry). The open reading frame (orf) for either protein is inserted at the targeted chromosomal location by λ Red recombination using an adjacent drug-resistance cassette ( or ) for selection. The drug-resistance gene is flanked by flippase (Flp) recognition target (FRT) sites in direct orientation, which allows removal of the cassette by Flp-mediated site-specific recombination once the construct is obtained, if desired. The method is specifically designed for the construction of translational fusions producing hybrid proteins with a fluorescent carboxyl-terminal domain. The fluorescent protein-encoding sequence can be placed at any codon position of the target gene's mRNA where the fusion produces a reliable reporter for gene expression. Internal and carboxyl-terminal fusions to sfGFP are suitable for studying protein localization in bacterial subcellular compartments.
我们描述了一种简单的基于重组酶的方法，用于将单拷贝基因融合到超折叠 GFP（sfGFP）和单体 Cherry（mCherry）中。通过使用相邻的药物抗性盒（或）进行选择，通过 λ Red 重组将蛋白质的开放阅读框（orf）插入到靶向的染色体位置。药物抗性基因两侧为正向定向的翻转酶（Flp）识别靶标（FRT）位点，如果需要，一旦获得构建体，Flp 介导的位点特异性重组可以去除盒。该方法专门用于构建产生带有荧光羧基末端结构域的杂种蛋白的翻译融合。荧光蛋白编码序列可以放置在目标基因 mRNA 的任何密码子位置，在该位置融合产生可靠的基因表达报告基因。sfGFP 的内部和羧基末端融合适用于研究细菌亚细胞区室中的蛋白质定位。
