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):663-670. doi: 10.1101/pdb.prot107951.
This protocol uses conditional plasmids carrying the open reading frame (orf) of either superfolder green fluorescent protein (sfGFP) or monomeric Cherry (mCherry) fused to a flippase (Flp) recognition target (FRT) site. In cells expressing the Flp enzyme, site-specific recombination between the plasmid-borne FRT and an FRT "scar" in a target gene in the bacterial chromosome results in chromosomal integration of the plasmid with the concomitant in-frame fusion of the target gene to the fluorescent protein orf. This event can be positively selected using an antibiotic-resistance marker ( or ) present on the plasmid. This method is slightly more laborious than generating the fusion directly by recombineering and has the limitation that the selectable marker is no longer removable. However, it has the advantage that it can be more readily integrated in mutational studies, allowing conversion of in-frame deletions resulting from Flp-mediated excision of a drug-resistance cassette (e.g., all those of the "Keio collection") into fluorescent protein fusions. Furthermore, in studies that require that the amino-terminal moiety of the hybrid protein keeps its biological activity, presence of the FRT "linker" sequence at the fusion junction makes it less likely for the fluorescent domain to sterically interfere with the folding of the amino-terminal domain.
本方案使用携带融合至弗林蛋白酶识别靶位（FRT）的超折叠绿色荧光蛋白（sfGFP）或单体 Cherry（mCherry）的开放阅读框（orf）的条件性质粒。在表达 Flp 酶的细胞中，质粒携带的 FRT 与细菌染色体中靶基因内的 FRT“疤痕”之间的特异性重组导致质粒与荧光蛋白orf 框内融合的染色体整合。此事件可通过质粒上存在的抗生素抗性标记（或）进行阳性选择。此方法比通过重组酶直接生成融合体稍微繁琐，且其局限性在于可选择的标记不再可移除。但是，它的优点是更易于在突变研究中进行整合，允许 Flp 介导的药物抗性盒（例如，“Keio 集合”中的所有盒）切除时产生的框内缺失转化为荧光蛋白融合体。此外，在需要保持杂交蛋白氨基末端部分生物活性的研究中，融合连接处的 FRT“接头”序列使荧光结构域更不可能因空间位阻而干扰氨基末端结构域的折叠。
