Construction of targeted single copy lac fusions using lambda Red and FLP-mediated site-specific recombination in...

A simple method for the construction of targeted transcriptional and translational fusions to the lac operon using FLP mediated site-specific recombination is described. Conditional plasmids containing promoterless lacZY genes and the FLP recognition target (FRT) site in both orientations were constructed for generating transcriptional fusions. Similarly, a plasmid used to create translational fusions was constructed in which the endogenous translational start of lacZ has been removed. These plasmids can be transformed into strains containing a single FRT site, which was previously integrated downstream of the promoter of interest using the lambda Red recombination method. The FLP protein produced from a helper plasmid that contains a conditional origin of replication promotes site-specific recombination between the FRT sites, resulting in an integrated lac fusion to the gene of interest. Transcriptional fusions to the Salmonella typhimurium genes sodCII and sitA were constructed using this method and shown to respond appropriately to mutations in the respective regulatory genes, rpoS and fur. Translational fusions were also constructed using this method. In this case, expression of beta-galactosidase was dependent on translation of the target protein. Given that the FLP recombinase does not require host factors for function and that this method requires no molecular cloning, this method should be applicable for the analysis of gene expression in a variety of organisms.