Measurement of Firefly Luciferase Reporter Gene Activity from Cells and Lysates Using Escherichia coli Arsenite and Mercury Sensors (original) (raw)
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Analytical Biochemistry, 2004
A cell-free biosensor for the detection of transcription induction by speciWc small-molecule ligands is presented. As model systems, tetracycline and mercury-inducible promoters were used containing WreXy luciferase as reporter gene. Escherichia coli S30 extract was prepared and used for coupled transcription-translation reactions. By using puriWed Tet repressor and MerR regulatory proteins, we could study repressor-operator interactions for optimizing the relative concentrations of each component. Previously, detection of tetracycline and mercury using similar transcriptional regulation in whole living cells has been carried out. As compared to whole-cell biosensors, our results showed better sensitivity for the detection of tetracycline and the toxic eVect of mercury was avoided in the cell-free system. Also, as the system omits cell cultivation and bacterial membranes as molecule passage inhibitors, it is possible to carry out assays in much shorter times and without the use of genetically modiWed organisms. 2004 Elsevier Inc. All rights reserved.
Environmental Challenges, 2000
Contamination of the environment by toxic compounds is a problem of global concern and in addressing this problem, it is necessary to identify the mechanisms by which specific agents exert their toxic effects, and develop effective, inexpensive strategies for detecting compounds in their biologically active and available form. We have used reporter gene fusion technology to identify genes, in the genetically well-characterized bacterium Escherichia coli, whose expression is affected by specific environmental toxins. As an added benefit of our approach, we have elaborated methods to use these gene fusion clones as biosensors to detect specific toxic agents, such as arsenic oxyanions. Arsenic is an abundant and useful element which is also an environmental toxin that can pose severe risks to health. Arsenic toxicity varies with oxidation state, organometallic form, and bioavailability. The Escherichia coli arsB fusion strains respond specifically to arsenic in its toxic, oxyanionic form, and can detect bioavailable amounts of these oxyanions in contaminated water samples. Combinations of different biosensor clones and assay automation will augment the use of luminescent biosensors for the detection of specific toxic agents in the environment.
Environmental Toxicology & Water Quality, 1990
A method for evaluation of toxicity of aqueous solutions is described based on the use of genetically engineered Escherichia coli. The genes encoding bacterial luciferase have been cloned from Vibrio harveyi to a deep rough mutant of E. coli under the control of the lac promoter. Light production by this strain has been stabilized by optimizing several parameters having an effect on the gene expression. Toxicity measurements were performed for selected metals and organic solvents to determine the sensitivity of the test strain. Effective concentrations calculated from these measurements show that this method has a sensitivity equal to other normally used methods. The test can be performed using buffers with low ionic strength without any significant change in the stability of the light emitted. Moreover, the method does not necessitate the use of special equipment or skills.
Current Opinion in Biotechnology, 2013
A wide variety of whole cell bioreporter and biosensor assays for arsenic detection has been developed over the past decade. The assays permit flexible detection instrumentation while maintaining excellent method of detection limits in the environmentally relevant range of 10-50 mg arsenite per L and below. New emerging trends focus on genetic rewiring of reporter cells and/or integration into microdevices for more optimal detection. A number of case studies have shown realistic field applicability of bioreporter assays.
2020
Background A bacterial biosensor refers to genetically engineered bacteria that produces an assessable signal in the presence of a physical or chemical agent in the environment. Methods We designed and evaluated a bacterial biosensor expressing a luciferase-reporting gene that is controlled by pbr and cadA promoters in Cupriavidus metallidurans (previously Ralstonia metallidurans) CH34 and pI258 plasmids of Staphylococcus aureus , respectively, for detection of heavy metals. In the present study, we produced biosensor plasmids designated pGL3-luc/pbrbiosensor and pGL3-luc/cad-biosensor, which were based on the expression of luc+ under the control of the cad promoter and the cadC gene of S. aureus plasmid pI258 and pbr promoter and pbrR gene from plasmid pMOL30 of Cupriavidus metallidurans. Results: We found that the biodegradable pGL3-luc/pbr-biosensor could measure lead concentrations between 1-100 μM in the presence of other metals such as zinc, cadmium, tin and nickel, in the presence of which gene expression of the reporter was not observed. The pGL3-luc/cad-biosensor was able to detect lead concentrations between 10 nM to 10 μM. Conclusions: This biosensor was found to be a specific sensor for identifying lead ions in both environmental and biological samples.
Background: A bacterial biosensor refers to genetically engineered bacteria that produce an assessable signal in the presence of a physical or chemical agent in the environment. Methods: We have designed and evaluated a bacterial biosensor expressing a luciferase-reporter gene controlled by pbr and cadA promoters in Cupriavidus metallidurans (previously termed Ralstonia metallidurans) containing the CH34 and pI258 plasmids of Staphylococcus aureus, respectively, and that can be used for the detection of heavy metals. In the present study, we have produced and evaluated biosensor plasmids designated pGL3-luc/pbr-biosensor and pGL3-luc/cad-biosensor, that were based on the expression of luc+ under the control of the cad promoter and the cadC gene of S. aureus plasmid pI258 and pbr promoter and pbrR gene from plasmid pMOL30 of Cupriavidus metallidurans. Results: We found that the pGL3-luc/pbr-biosensor may be used to measure lead concentrations between 1-100 μM in the presence of other...
Biosensors and Bioelectronics, 2014
Genetically engineered microbial biosensors have yet to realize commercial success in environmental applications due, in part, to difficulties associated with transducing and transmitting traditional bioluminescent information. Bioelectrochemical systems (BESs) output a direct electric signal that can be incorporated into devices for remote environmental monitoring. Here, we describe a BES-based biosensor with genetically encoded specificity for a toxic metal. By placing an essential component of the metal reduction (Mtr) pathway of Shewanella oneidensis under the control of an arsenic-sensitive promoter, we have genetically engineered a strain that produces increased current in response to arsenic when inoculated into a BES. Our BES-based biosensor has a detection limit of 40 μM arsenite with a linear range up to 100 μM arsenite. Because our transcriptional circuit relies on the activation of a single promoter, similar sensing systems may be developed to detect other analytes by the swap of a single genetic part.
1996
In the quantitation of gene expression using firefly luciferase, a second reporter gene is commonly used to minimize experimental variability. However, traditional co-reporters (e.g., CAT, beta-Gal or GUS) are inconvenient due to differences in their respective assay chemistries, handling requirements and measurement characteristics. Promega introduces a superior dual-reporter technology integrating the assay of firefly luciferase with the Renilla luciferase assay. The Dual-LuciferaseTM Reporter Assay System*, in combination with the pRL Vectors which express Renilla luciferase as the second reporter, offers the exceptional speed, sensitivity and convenience of two luciferase reporter assays in a single-tube format. The system also includes Passive Lysis Buffer, formulated to provide quantitative solubilization of both luciferases from mammalian cells cultured in multi-well plates without the need for individual processing of each sample. The Dual-LuciferaseTM Reporter Assay System ...
Response Characteristics of Arsenic-Sensitive Bioreporters Expressing the gfp Reporter Gene
Microchimica Acta, 2005
This paper describes the development of an analytical technique for arsenic analyses that is based on genetically-modified bioreporter bacteria bearing a gene encoding for the production of a green fluorescent protein (gfp). Upon exposure to arsenic (in the aqueous form of arsenite), the bioreporter production of the fluorescent reporter molecule is monitored spectroscopically. We compared the response measured as a function of time and concentration by steady-state fluorimetry (SSF) to that measured by epi-fluorescent microscopy (EFM). SSF is a bulk technique; as such it inherently yields less information, whereas EFM monitors the response of many individual cells simultaneously and data can be processed in terms of population averages or subpopulations. For the bioreporter strain used here, as well as for the literature we cite, the two techniques exhibit similar performance characteristics. The results presented here show that the EFM technique can compete with SSF and shows substantially more promise for future improvement; it is a matter of research interest to develop optimized methods of EFM image analysis and statistical data treatment. EFM is a conduit for understanding the dynamics of individual cell response vs. population response, which is not only a matter of research interest, but is also promising in the practical terms of developing micro-scale analysis.