title: Cell damage detection using Escherichia coli reporter plasmids: fluorescent and colorimetric assays.
abstract: Bacterial reporter assays are powerful tools used to study the effect of different compounds that affect the physiology of cellular processes. Most bacterial reporters are luciferase based and can be monitored in real time. In the present study we designed and implemented two sets of Escherichia coli bacterial reporter assays, using a multicopy plasmid system. Each reporter strain was constructed using either green fluorescent protein or β-galactosidase (LacZ) proteins. The designed reporter strains are capable of responding in a specific manner to molecules that either oxidative stress, or membrane, protein, or DNA damage. In order to respond to the desired stimulus, promoter sequences from E. coli were used. These sequences correspond to the promoter of the major catalase (KatG) activated with cellular oxidative damage, the promoter of the β-hydroxydecanoyl-ACP dehydrase (FabA) which is activated with membrane perturbation, the promoter of DNA recombinase (RecA) which is activated by DNA lesions. For protein misfolding, the promoter of the heat-shock responsive chaperon (DnaK) was used. Our constructs displayed activation to damage from specific stimuli, and low response to nonspecific stimuli was detected. Our results suggest that these types of bacterial reporter strains can be used in semiquantitative (fluorometric) and qualitative (β-galactosidase activity) studies of different xenobiotic substances and pollutants.
authors.affiliation: División de Ciencias Naturales y Exactas, Departamento de Biología, Universidad de Guanajuato, Noria Alta s/n, 36050, Guanajuato, Gto., Mexico.
authors.forename: Felipe
authors.initials: F
authors.lastname: Padilla-Martínez
authors.forename: Luz Adriana
authors.initials: LA
authors.lastname: Carrizosa-Villegas
authors.forename: Ángeles
authors.initials: Á
authors.lastname: Rangel-Serrano
authors.forename: Itzel
authors.initials: I
authors.lastname: Paramo-Pérez
authors.forename: Verónica
authors.initials: V
authors.lastname: Mondragón-Jaimes
authors.forename: Fernando
authors.initials: F
authors.lastname: Anaya-Velázquez
authors.forename: Felipe
authors.initials: F
authors.lastname: Padilla-Vaca
authors.forename: Bernardo
authors.initials: B
authors.lastname: Franco
mesh_terms: D001483:Base Sequence; D015374:Biosensing Techniques; D003124:Colorimetry / Q000379:methods*; D004249:DNA Damage / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D018384:Oxidative Stress / Q000502:physiology; D010957:Plasmids; D011401:Promoter Regions, Genetic; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D049452:Green Fluorescent Proteins; D001616:beta-Galactosidase