title: A cloning vector for creation of Escherichia coli lacZ translational fusions and generation of linear template for chromosomal integration.
abstract: A novel cloning vector to aid in the construction of single copy β-galactosidase reporter systems for gene expression studies in lactose metabolizing Escherichia coli strains, including STEC, is described. The plasmid allows construction of translational fusions of cloned gene promoters to a short segment of E. coli lacZ. A selectable spectinomycin resistance marker flanked by a short lacI segment is positioned 5' to the cloning site. PCR amplification using opposing primers complementary to the upstream lacI fragment and the downstream lacZ fragment generates a linear template suitable for integration using pRedET recombination. Integration of linear template derived from the recombinant plasmid into host strains replaces the entire native lacZ promoter and fuses the promoter of interest in-frame with the lacZ gene, thus simultaneously producing a single-copy, chromosomal reporter system and eliminating background lacZ expression. Studies comparing ahpC expression from a chromosomal fusion in the lac open with that on a plasmid in E. coli strain EDL933 are shown.
authors.affiliation: Molecular Characterization of Foodborne Pathogens Research Unit, Eastern Regional Research Center, Agricultural Research Service, US Department of Agriculture, Wyndmoor, PA 19038, USA. gaylen.uhlich@ars.usda.gov
authors.forename: Gaylen A
authors.initials: GA
authors.lastname: Uhlich
authors.forename: Chin-Yi
authors.initials: CY
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D002876:Chromosomes, Bacterial / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors / Q000235:genetics*; D007763:Lac Operon / Q000235:genetics*; D007785:Lactose / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics; D000198:Spectinomycin; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D000198:Spectinomycin; D001616:beta-Galactosidase; D007785:Lactose