Cell damage detection using Escherichia coli reporter plasmids: fluorescent and colorimetric assays
- PDF / 963,333 Bytes
- 7 Pages / 595.276 x 790.866 pts Page_size
- 60 Downloads / 181 Views
ORIGINAL PAPER
Cell damage detection using Escherichia coli reporter plasmids: fluorescent and colorimetric assays Felipe Padilla‑Martínez1 · Luz Adriana Carrizosa‑Villegas1 · Ángeles Rangel‑Serrano1 · Itzel Paramo‑Pérez1 · Verónica Mondragón‑Jaimes2 · Fernando Anaya‑Velázquez1 · Felipe Padilla‑Vaca1 · Bernardo Franco1
Received: 17 September 2014 / Revised: 21 December 2014 / Accepted: 7 May 2015 © Springer-Verlag Berlin Heidelberg 2015
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 Communicated by Erko Stackebrandt. * Bernardo Franco [email protected] 1
División de Ciencias Naturales y Exactas, Departamento de Biología, Universidad de Guanajuato, Noria Alta s/n, 36050 Guanajuato, Gto., Mexico
2
Unidad Académica de Ciencias Químico Biológicas y Farmacéuticas, Universidad Autónoma de Nayarit, Ciudad de la Cultura “Amado Nervo”, C.P. 63155 Tepic, Nayarit, Mexico
be used in semiquantitative (fluorometric) and qualitative (β-galactosidase activity) studies of different xenobiotic substances and pollutants. Keywords Cellular damage · DNA damage · Escherichia coli · β-Galactosidase · Green fluorescent protein · Membrane damage · Oxidative damage · Protein damage
Introduction Since the emergence of recombinant DNA technology, scientists have being able to achieve the control of transcriptional regulatory circuits and gene expression, modifying organisms to respond to environmental changes. Wholecell reporter assays have become powerful tools to address important toxicological, environmental and physiological questions (Raut et al. 2012). They can shed light on the primary action mechanism of antibiotics, anti-microbial compounds and xenobiotics. Likewise, whole-cell reporter assays can help evaluate the toxicity of several chemicals and the fraction of these pollutants
Data Loading...
