Construction of Single-Copy Fluorescent Protein Fusions by One-Step Recombineering

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 (kan or cat) 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.