Characterization and optimization of two methods in the immobilization of 12 bioluminescent strains.
ABSTRACT Twelve recombinant bioluminescent bacteria have been immobilized within the wells of a 96-well plate using two different matrices--agar and sol-gel. All 12 strains were immobilized within individual wells of the plates and the sensitivity of the strains and the stability of the responses were determined for select chemicals. Each strain was exposed to seven well-characterized chemicals over a wide range of concentrations to demonstrate their individual selectivity for specific toxicants. Although the sensitivity of the immobilized cells was generally lower than cultures grown in liquid media, they were comparable. For example, strain DPD1710, which responds to DNA damage was able to detect mitomycin C, a genotoxin, at a minimum concentration of 0.6 ppb. When immobilized, the lower limit of detection was between 1 and 10 ppb. Finally, using compounds that are known to elicit a response from each of the strains, the stability of the bioluminescent responses were measured over an extended period of 4 weeks. Although the activity of several strains decreased over time, the majority of the strains used in both immobilized systems were still responsive.
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ABSTRACT: Aberrations in the growth and transcriptome of Escherichia coli str. BL21(DE3) were determined when exposed to varying concentrations of ferulic acid (0.25-1 g/L), an aromatic carboxylic acid identified within lignin-cellulose hydrolysate samples. The expression of several individual genes (aaeA, aaeB, inaA and marA) was significantly induced, i.e., more than 4-fold, and thus these genes and the heat shock response gene htpG were selected as biomarkers to monitor E. coli's responses to five additional hydrolysate-related compounds, including vanillic acid, coumaric acid, 4-hydroxybenzoic acid, ferulaldehyde and furfural. While all of the biomarkers showed dose-dependent responses to most of the compounds, expression of aaeA and aaeB showed the greatest induction (5-30-fold) for all compounds tested except furfural. Lastly, the marA, inaA and htpG genes all showed higher expression levels when the culture was exposed to spruce hydrolysate samples, demonstrating the potential use of these genes as biomarkers.Bioresource Technology 02/2012; 114:450-6. DOI:10.1016/j.biortech.2012.02.085 · 5.04 Impact Factor
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ABSTRACT: This chapter deals with the use of bioluminescent microorganisms in environmental monitoring, particularly in the assessment of the ecotoxicity of pollutants. Toxicity bioassays based on bioluminescent microorganisms are an interesting complement to classical toxicity assays, providing easiness of use, rapid response, mass production, and cost effectiveness. A description of the characteristics and main environmental applications in ecotoxicity testing of naturally bioluminescent microorganisms, covering bacteria and eukaryotes such as fungi and dinoglagellates, is reported in this chapter. The main features and applications of a wide variety of recombinant bioluminescent microorganisms, both prokaryotic and eukaryotic, are also summarized and critically considered. Quantitative structure-activity relationship models and hormesis are two important concepts in ecotoxicology; bioluminescent microorganisms have played a pivotal role in their development. As pollutants usually occur in complex mixtures in the environment, the use of both natural and recombinant bioluminescent microorganisms to assess mixture toxicity has been discussed. The main information has been summarized in tables, allowing quick consultation of the variety of luminescent organisms, bioluminescence gene systems, commercially available bioluminescent tests, environmental applications, and relevant references.
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ABSTRACT: Recombinant yeast assays (RYAs) constitute a suitable tool for the environmental monitoring of compounds with endocrine disrupting activities, notably estrogenicity and androgenicity. Conventional procedures require yeast reconstitution from frozen stock, which usually takes several days and demands additional equipment. With the aim of applying such assays to field studies and making them more accessible to less well-equipped laboratories, we have optimized RYA by the immobilization of Saccharomyces cerevisiae cells in three different polymer matrices - gelatin, Bacto agar, and Yeast Extract Peptone Dextrose agar - to obtain a ready-to-use version for the fast assessment of estrogenic and androgenic potencies of compounds and environmental samples. Among the three matrices, gelatin showed the best results for both testosterone (androgen receptor yeast strain; AR-RYA) and 17β-estradiol (estrogen receptor yeast strain; ER-RYA). AR-RYA was characterized by a lowest observed effect concentration (LOEC), EC50 and induction factor (IF) of 1nM, 2.2nM and 51, respectively. The values characterizing ER-RYA were 0.4nM, 1.8nM, and 63, respectively. Gelatin immobilization retained yeast viability and sensitivity for more than 90d of storage at 4°C. The use of the immobilized yeast reduced the assay duration to only 3h without necessity of sterile conditions. Because immobilized RYA can be performed either in multiwell microplates or glass tubes, it allows multiple samples to be tested at once, and easy adaptation to existing portable devices for direct in-field applications. Copyright © 2015 Elsevier Ltd. All rights reserved.Chemosphere 03/2015; 132:56-62. DOI:10.1016/j.chemosphere.2015.02.063 · 3.50 Impact Factor