Application of promoter swapping techniques to control expression of chromosomal genes

Abstract

The ability to control the expression of chromosomal genes is important for many applications, including metabolic engineering and the functional analysis of cellular processes. This mini-review presents recent work on the application of techniques that allow researchers to replace a chromosomal promoter with one designed for a specific level of activity, thereby exerting precise transcriptional control while retaining the natural genetic context of a gene or operon. This technique, termed promoter swapping, involves the creation of a PCR product that encodes a removable antibiotic resistance cassette and an engineered promoter. Short homology sequences on the ends of the PCR fragment target it for homologous recombination with the chromosome catalyzed by phage-derived recombination proteins. After the PCR product is introduced by electroporation into an appropriate acceptor strain, antibiotic resistance selects the desired recombination products. The antibiotic resistance cassette is then removed from the strain by site-specific recombination leaving the engineered promoter precisely positioned upstream of a target gene but downstream of a short scar consisting of a single site-specific recombination site.