Dataset Information

Internal arsenite bioassay calibration using multiple bioreporter cell lines.

ABSTRACT: Bioassays with bioreporter bacteria are usually calibrated with analyte solutions of known concentrations that are analysed along with the samples of interest. This is done as bioreporter output (the intensity of light, fluorescence or colour) does not only depend on the target concentration, but also on the incubation time and physiological activity of the cells in the assay. Comparing the bioreporter output with standardized colour tables in the field seems rather difficult and error-prone. A new approach to control assay variations and improve application ease could be an internal calibration based on the use of multiple bioreporter cell lines with drastically different reporter protein outputs at a given analyte concentration. To test this concept, different Escherichia coli-based bioreporter strains expressing either cytochrome c peroxidase (CCP, or CCP mutants) or ?-galactosidase upon induction with arsenite were constructed. The reporter strains differed either in the catalytic activity of the reporter protein (for CCP) or in the rates of reporter protein synthesis (for ?-galactosidase), which, indeed, resulted in output signals with different intensities at the same arsenite concentration. Hence, it was possible to use combinations of these cell lines to define arsenite concentration ranges at which none, one or more cell lines gave qualitative (yes/no) visible signals that were relatively independent of incubation time or bioreporter activity. The discriminated concentration ranges would fit very well with the current permissive (e.g. World Health Organization) levels of arsenite in drinking water (10 µg l(-1)).

SUBMITTER: Wackwitz A

PROVIDER: S-EPMC3864448 | biostudies-literature | 2008 Mar

REPOSITORIES: biostudies-literature

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Internal arsenite bioassay calibration using multiple bioreporter cell lines.

Wackwitz Anke A Harms Hauke H Chatzinotas Antonis A Breuer Uta U Vogne Christelle C Van Der Meer Jan Roelof JR

Microbial biotechnology 20080301 2

Bioassays with bioreporter bacteria are usually calibrated with analyte solutions of known concentrations that are analysed along with the samples of interest. This is done as bioreporter output (the intensity of light, fluorescence or colour) does not only depend on the target concentration, but also on the incubation time and physiological activity of the cells in the assay. Comparing the bioreporter output with standardized colour tables in the field seems rather difficult and error-prone. A ...[more]

PMID: 21261832

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Project description:Despite various efforts to develop tools to detect and compare the activity and catabolic potential and activity for pollutant degradation of microorganisms in environmental samples, an open-source, curated and reliable method is still required. Here we report on a customize normalization system that can be applied to any microarray design, allowing the assessment of reliability of signals and enabling cross-experiment comparisons. Probes for the underlying catabolic gene array were designed based on manually curated databases for catabolic key genes. Signals were assigned to the respective catabolic protein subfamily that allows to inferring the functions and substrate specificity. The placement of probes for critical degradation nodes and subsequent metabolic steps allows the retrieval of information on the metabolic net. Information on gene localization from genome surveys was correlated to signals of putative hosts to generate phylogenetic community information. Hence, this novel array system was validated using genomic DNA of genome sequenced bacterial strains hosting biodegradation functions and applied to genomic DNA and RNA extracted from environmental samples under aromatic pollution pressure. Catabolic microarray was optimized for detection and expression of catabolic genes involved in the degradation of pollutant compounds using genome genomic DNA of type strains: Burkholderia xenovorans LB400, Cupriavidus necator JMP134, Pseudomonas putida F1 and Sphingomonas wittichii RW1. Afterwards, this microarray was applied for environmental samples. For microcosm experiments, 200 ml of each groundwater were incubated in closed 1 l Erlenmeyer flasks at 20 degree C and 50 rpm. Second sets of 200 ml microcosms were supplemented with naphthalene (100 mg), which had above been identified as a contaminant present in both groundwaters in similar amounts. Naphthalene was supplied via the vapor phase and incubated under identical conditions as the control microcosms. After 4 days of incubations, 50 ml of each of the 4 microcosms were subjected to genomic DNA extraction and another 50 ml to RNA extraction.

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Internal arsenite bioassay calibration using multiple bioreporter cell lines.

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Internal arsenite bioassay calibration using multiple bioreporter cell lines.

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