A comprehensive toolbox for the rapid construction of lacZ fusion reporters

beta-Galactosidase encoded by lacZ remains a popular reporter enzyme. Here, we present three fast and convenient tools that facilitate rapid construction of reporter lacZ fusions. The first enables the simple generation of lacZ (slacZ)-based chromosomally encoded reporter fusions within the lac operon in Escherichia coli using Red (R)/ET (R) recombination. The slacZ tool is based on rpsL counter-selection in combination with homologous recombination catalyzed by the X Red recombinase, and blue/white screening. This permits construction of transcriptional and translational reporter lacZ fusions within a day. The second tool allows the introduction of lacZ reporter fusions into the chromosome by a single-crossover method. The strategy relies on the gamma-origin-based suicide vector pNPTS138-R6KT, which can only replicate in lambda pir E. coli strains. The third tool comprises four pBBR1-based broad-host-range vectors for transcriptional and translational lacZ fusions. The functionality of our toolbox was confirmed by the K+-dependent activation of kdp promoter-lacZ fusions in vivo. (c) 2012 Elsevier B.V. All rights reserved.