Converting an FRT-Tagged Gene into a Fluorescent Protein Gene Fusion by Flp-Mediated Site-Specific Recombination.

Abstract

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 (kan or cat) 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.