title: A comprehensive toolbox for the rapid construction of lacZ fusion reporters.
abstract: β-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®/ET® recombination. The slacZ tool is based on rpsL counter-selection in combination with homologous recombination catalyzed by the λ 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 γ-origin-based suicide vector pNPTS138-R6KT, which can only replicate in λpir 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.
authors.affiliation: Munich Center for Integrated Protein Science (CIPSM) at the Department of Microbiology, Ludwig-Maximilians-Universität München, 82152 Martinsried, Germany.
authors.forename: Luitpold
authors.initials: L
authors.lastname: Fried
authors.forename: Jürgen
authors.initials: J
authors.lastname: Lassak
authors.forename: Kirsten
authors.initials: K
authors.lastname: Jung
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D007763:Lac Operon; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D000091243:Ribosomal Protein S9; D001616:beta-Galactosidase / Q000235:genetics* / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D000091243:Ribosomal Protein S9; C000717190:RpsI protein, E coli; D001616:beta-Galactosidase