DOCSLIB.ORG

A Bioluminescent Yeast-Reporter System For

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text

Load more

A Bioluminescent Yeast-Reporter System for Screening Chemicals for Estrogenic Effects John Sanseverino, The Center for Environmental Biotechnology The University of Tennessee Abstract Saccharomyces cerevisiae BLYES The Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) created by the Environmental Protection Agency was mandated with developing methods to screen PGK GPD ADH1 ERE luxA approximately 87,000 chemicals for biological effects on estrogen, androgen and thyroid luxC ERE luxE IRES hormone systems. As part of this mandate, EDSTAC proposed that EPA develop rapid, high IRES IRES pBLYES luxD pLCIRESDE- frp throughput screening systems to assess a compound’s effects on hormonal systems. The ~10kb luxB IRESfrp Center for Environmental Biotechnology at the University of Tennessee has re-engineered ~12.1kb the Saccharomyces cerevisiae YES colorimetric estrogen reporter system (Routledge and Sumpter, 1996) to produce bioluminescence in response to estrogen or environmental A B estrogens (S. cerevisiae BLYES; Figure 1; Gupta et al., 2003). Bioluminescence is a Luciferase FMNH reagentless system eliminating the need for expensive chromophores. Light detection is 2 C D E more sensitive than absorbance detection thus shortening the development time of the assay. Light FMN 490nm In previous work, strain BLYES was exposed to the estrogenic compounds 17β-estradiol, Myristyl aldehyde 17α-estradiol, 17α-ethynyl estradiol, estrone, and 3,4′,5-trichloro-4-biphenylol and 2 O compared to the YES assay (Fig. 2). The EC values correlated linearly (R =0.97) between 2 50 Myristic acid the 2 assays. Sensitivities of both assays decreased in the order 17β-estradiol > 17α-ethynyl H O - estradiol > estrone > 17α-estradiol, with no significant response generated from 3,4′,5- 2 trichloro-4-biphenylol where the hydroxyl group is the stericly hindered by the paired ortho chlorines. The BLYES screen consistently detected estrogenic potencies at 5- to 10-fold lower levels than those attained in the YES assay. Moreover, bioluminescence was Figure. 1. Construction of the estrogen inducible bioreporter S. cerevisiae BLYES. detectable in less than 4 hours as compared to 3 days for the colorimetric YES strain. Synthesis of luxA is regulated on plasmid pEREAB by upstream incorporation of two The primary objective of this research is to validate the BLYES system and develop a sequential estrogen response elements (ERE) coupled to a human phosphoglycerate kinase standard operating procedure for routine chemical analysis. Work is in progress to test the (PGK) promoter. The luxB component of the luciferase is supplied via independent S. cerevisiae BLYES using the proposed 78 substances (ICCVAM, 2002) listed for expression from a fused IRES. The luxC, luxD, luxE and frp genes are under control of validation of estrogen receptors and correlate to the colorimetric S. cerevisiae YES assay. constitutive promoters on the plasmid pLCDEfrp. The human estrogen receptor (hER-α) is Parallel research includes developing the S. cerevisiae BLYES into a standard assay suitable inserted in the S. cerevisiae chromosome. for HTS of chemicals and to modify the lux genes for optimum transcription/translation in S. cerevisiae thus increasing sensitivity of the assay. Goals The purpose of Tier 1 screening is to identify substances that have the potential to interact 200 ) 17β-estradiol (R2 = 0.98) with the endocrine system. The colorimetric-based YES assay has been widely used in the 600 180 17α-ethynyl estradiol (R2 = 0.99) literature and this laboratory and is a very useful tool for assessing estrogenicity of a estrone (R2 = 0.99) 160 17α-estradiol (R2 = 0.98) compound or environmental sample. Development of the bioluminescent version of the 140 YES system (S. cerevisiae BLYES) has the potential to enhance the utility of this system. 120 The primary objective of this research is to (i) validate the BLYES system and develop a standard operating procedure for routine chemical analysis; and (ii) develop an androgen 100 bioluminescent reporter system analogous to the BLYES system. 80 -7.6 -7.8 y = 0.78x - 1.03 2 The specific tasks of this research are: 60 R = 0.99 -8.0 40 -8.2 1. Test the S. cerevisiae BLYES using the proposed 78 substances (see ICCVAM, 2002) -8.4 20 log [BLYES] (M) listed for validation of estrogen receptors and correlate to the colorimetric S. cerevisiae -8.6 YES assay. 0 -8.8 -10.0 -9.8 -9.6 -9.4 -9.2 -9.0 -8.8 -8.6 -8.4 Normalized Bioluminescence (cps/OD Bioluminescence Normalized log [YES] (M) 2. Develop the S. cerevisiae BLYES into a standard assay suitable for HTS of chemicals. 10-11 10-10 10-9 10-8 10-7 10-6 10-5 Concentration (M) 3. Modify plasmid pEREAB construct for improved sensitivity. 4. Develop a yeast-based reporter for the detection of androgens. Figure 2. EC50 dose response profiles of the S. cerevisiae BLYES bioreporter to the estrogenic compounds 17β-estradiol (z), 17α-ethynyl estradiol (V), estrone (), and 17α-estradiol (); (n = 4). Inset: EC50 dose response correlations between the lacZ based YES and lux based BLYES estrogenic assays. 70,000 Impact 60,000 50,000 This EPA-sponsored research seeks to standardize a bioluminescent yeast assay for screening substances that potentially interact with the human estrogen receptor. The 40,000 bioluminescent reporter system has several advantages including: 30,000 • A low-cost, reagentless reporter system that eliminates the extra manipulation and cost of adding exogenous reagents. Bioluminescence 20,000 • Speed; preliminary data using 17β-estradiol indicated a bioluminescent response 10,000 in 2-4 hours and a maximum response in 15 hours (Fig. 3). 0 62.5 0 3 6 • Amenable to automated, high throughput analysis, data collection and 9 12 ppb interpretation. 15 18 21 24 0 27 Hours 30 o 33 • Cells for the assay can be prepared fresh or stored at –80 C. 36 • No animals are used in this assay. Figure 3. 3-Dimensional plot of bioluminescence vs. time for 0-62.5 ppb 17β-estradiol. Initial bioluminescence is observed in as little as 2 hours and peaks at approximately 15 h. • Elimination of labor-intensive cell culture assays. Acknowledgements Literature Cited Funds for this research are provided by EPA STAR Grant R-831302 and Gupta, R.K., S.S. Patterson, S. Ripp, M.L. Simpson, and G.S. Sayler. 2003. Expression of the The Center for Environmental Biotechnology at The University of Photorhabdus luminescens lux genes (luxA, B, C, D, and E) in Saccharomyces cerevisiae. FEMS Yeast Research. 4:305-313. Tennessee. Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM). 2002. Expert Principle Investigators/Contact Information Panel Evaluation on the Validation Status of In Vitro Test Methods for Detecting Endocrine Disruptors: Gary Sayler, T. Wayne Schultz, Alice Layton, John Sanseverino, Leslie Estrogen Receptor and Androgen Receptor Binding and Transcription Activation Assays. Expert Panel Final Report. Saidak, James Easter, Victoria Garrett. The Center for Environmental Biotechnology, The University of Tennessee 676 Dabney Hall, Routledge, E.J., and J.P. Sumpter. 1996. Estrogenic activity of surfactants and some of their degradation Knoxville, Tn 37996 products assessed using a recombinant yeast screen. Environ. Toxic. Chem. 15(3):241-248..
Recommended publications
  • Construction of a Copper Bioreporter Screening, Characterization and Genetic Improvement of Copper-Sensitive Bacteria

    Construction of a Copper Bioreporter Screening, Characterization and Genetic Improvement of Copper-Sensitive Bacteria

    Construction of a Copper Bioreporter Screening, characterization and genetic improvement of copper-sensitive bacteria Puria Motamed Fath This thesis comprises 30 ECTS credits and is a compulsory part in the Master of Science With a Major in Industrial Biotechnology, 120 ECTS credits No. 9/2009 Construction of a Copper Bioreporter-Screening, characterization and genetic improvement of copper-sensitive bacteria Puria Motamed Fath, [email protected] Master thesis Subject Category: Technology University College of Borås School of Engineering SE-501 90 BORÅS Telephone +46 033 435 4640 Examiner: Dr. Elisabeth Feuk-lagerstedt Supervisor, name: Dr. Saman Hosseinkhani Supervisor, address: Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University Tehran, Iran Date: 2009-12-14 Keywords: Copper Bioreporter, Luciferase assay, COP operon, pGL3, E. Coli BL-21 ii Dedicated to my parents to whom I owe and feel the deepest gratitude iii Acknowledgment I would like to express my best appreciation to my supervisor Dr. S. Hosseinkhani for technical supports, and my examiner Dr. E. Feuk-lagerstedt for her kind attention, also I want to thank Mr. A. Emamzadeh, Miss. M. Nazari, and other students of Biochemistry laboratory of Tarbiat Modares University for their kind corporations. iv Abstract In the nature, lots of organism applies different kinds of lights such as flourescence or luminescence for some purposes such as defense or hunting. Firefly luciferase and Bacterial luciferase are the most famous ones which have been used to design Biosensors or Bioreporters in recent decades. Their applications are so extensive from detecting pollutions in the environment to medical and treatment usages.
  • New Naphthalene Whole-Cell Bioreporter for Measuring and Assessing Naphthalene in Polycyclic Aromatic Hydrocarbons Contaminated Site

    New Naphthalene Whole-Cell Bioreporter for Measuring and Assessing Naphthalene in Polycyclic Aromatic Hydrocarbons Contaminated Site

    Accepted Manuscript New naphthalene whole-cell bioreporter for measuring and assessing naphthalene in polycyclic aromatic hydrocarbons contaminated site Yujiao Sun, Xiaohui Zhao, Dayi Zhang, Aizhong Ding, Cheng Chen, Wei E. Huang, Huichun Zhang PII: S0045-6535(17)31248-1 DOI: 10.1016/j.chemosphere.2017.08.027 Reference: CHEM 19725 To appear in: ECSN Received Date: 17 April 2017 Revised Date: 22 July 2017 Accepted Date: 7 August 2017 Please cite this article as: Sun, Y., Zhao, X., Zhang, D., Ding, A., Chen, C., Huang, W.E., Zhang, H., New naphthalene whole-cell bioreporter for measuring and assessing naphthalene in polycyclic aromatic hydrocarbons contaminated site, Chemosphere (2017), doi: 10.1016/j.chemosphere.2017.08.027. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ACCEPTED MANUSCRIPT 1 New naphthalene whole-cell bioreporter for measuring and assessing 2 naphthalene in polycyclic aromatic hydrocarbons contaminated site 3 Yujiao Sun a, Xiaohui Zhao a,b* , Dayi Zhang c, Aizhong Ding a, Cheng Chen a, Wei E. 4 Huang d, Huichun Zhang a. 5 a College of Water Sciences, Beijing Normal University, Beijing, 100875, PR China 6 b Department of Water Environment, China Institute of Water Resources and 7 Hydropower Research, Beijing, 100038, China 8 c Lancaster Environment Centre, Lancaster University, Lancaster, LA1 4YQ, UK 9 d Kroto Research Institute, University of Sheffield, Sheffield, S3 7HQ, United 10 Kingdom 11 12 Corresponding author 13 Dr Xiaohui Zhao 14 a College of Water Sciences, Beijing Normal UniversiMANUSCRIPTty, Beijing 100875, P.
  • Microbial Biodegradation and Bioremediation

    Microbial Biodegradation and Bioremediation

    5 Microbial Indicators for Monitoring Pollution and Bioremediation Tingting Xua, Nicole Perryb, Archana Chuahana, Gary Saylera and Steven Rippa aThe University of Tennessee, The Center for Environmental Biotechnology, The Joint Institute for Biological Sciences, Knoxville, TN, bDepartment of Biology, Wittenberg University, Springfield, OH 5.1 Introduction Microorganisms metabolically rely upon chemicals scavenged from their environ- ment and correspondingly defend themselves against toxic chemicals to ensure their growth and survival. These essential needs can be genetically exploited to create bioreporter organisms that can rapidly and specifically indicate the presence and bio- availability of target environmental contaminants. Using the toolbox of genetic engi- neering, genes or their operon units that are activated upon exposure to a target chemical agent or chemical class can be isolated and linked to reporter genes to serve as operational switches that bioindicate chemical presence with sensitivity and reproducibility that oftentimes rivals conventional analytical methods. Reporter genes most often consist of light signaling elements such as the bioluminescent bac- terial (lux) and firefly (luc) luciferases, green fluorescent protein (gfp as well as its palette of multicolored fluorescent derivatives), and colorimetric indicators like β-galactosidase (lacZ)(Close et al., 2009). These reporter genes are widely available within numerous types of cloning vectors for straightforward manipulation and have been successfully applied toward the creation of a large number of bioreporter organisms. The challenge in bioreporter development, however, lies in discovering and isolating the appropriate promoter gene switch that activates in response to the desired chemical target or targets. Although often provided via the evolutionary adaptation of microbial populations to new chemical bioavailability, particular pro- moter elements still must be isolated and characterized, which requires significant time, effort, and cost.
  • Redesign of Calcium-Regulated Protein Aequorin Towards the Development

    Redesign of Calcium-Regulated Protein Aequorin Towards the Development

    Redesign of calcium-regulated protein aequorin towards the development of a novel ion bioreporter A Thesis submitted to University College London for the degree of Doctor of Philosophy by Evlampia-Kyriaki Dimitriadou Department of Biochemical Engineering University College London UCL April 2014 I, Evlampia-Kyriaki Dimitriadou confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. - 2 - I dedicate this work to my loving parents, Sophia and Panagiotis - 3 - Abstract This thesis aimed to design novel sensor proteins that can identify and measure various metal ions in vivo and in situ . Metal ions play key role in the metabolism of the cell, and monitoring of calcium has helped interrogate cellular processes such as fertilisation, contraction and apoptosis. Real-time monitoring of more divalent metal ions like zinc and copper is required to gain much needed insight into brain function and associated disorders, such as Alzheimer’s and Parkinson’s disease. Aequorin is a calcium-regulated photoprotein originally isolated from the jellyfish Aequorea victoria . Due to its high sensitivity to calcium and its non-invasive nature, aequorin has been used as a real-time indicator of calcium ions in biological systems for more than forty years. The protein complex consists of the polypeptide chain apoaequorin and a tightly bound chromophore (coelenterazine). Trace amounts of calcium ions trigger conformational changes in the protein, which in turn facilitate the intermolecular oxidation of coelenterazine and concomitant production of CO 2 and a flash of blue light.
  • Bioassays and Bioreporters

    Bioassays and Bioreporters

    OECD Workshop on Environmental Biotechnology Rimini, Italy Bioassays and bioreporters Davide Merulla Jan Roelof van der Meer Thursday, September 16, 2010 Outline of the presentation • Current state of the art in use of bioassays • New approaches - promises, advantages • Case study - arsenic bioreporter assays • Implementation - hurdles, difficulties • The way forward? 2 OECD Workshop on Environmental Biotechnoogy Sept 17, 2010 Bioassays • Why use bioassays when there is beautiful, precise and expensive chemical analytics? Pictures: FACEiT consortium 3 OECD Workshop on Environmental Biotechnoogy Sept 17, 2010 Why bioassays? Simple reason: Measuring heart beat rates of a crab Picture: Ceri Lewis, Tamara Galloway • Chemical analysis can University of Exeter, UK measure total concentrations • Very difficult to predict on the basis of chemistry: – Concentrations experienced by living organisms – Biological effects of single compounds – Effects of mixtures – Acute - chronic effects 4 OECD Workshop on Environmental Biotechnoogy Sept 17, 2010 Bioassays come in different forms • In situ examination of exposed organisms • Exposure of model organisms under standardized conditions • Use of cell cultures, single cell models • Use of genetically engineered single cell models • biosensors with purified enzymes or biological components 5 OECD Workshop on Environmental Biotechnoogy Sept 17, 2010 1) In situ examination of exposed organisms MSC Napoli, Plymouth January 2007 EXAMPLE • Local limpets found on shores in- and outside the presumed contaminated
  • Genetically Modified Whole-Cell Bioreporters for Environmental

    Genetically Modified Whole-Cell Bioreporters for Environmental

    Ecological Indicators 28 (2013) 125–141 Contents lists available at SciVerse ScienceDirect Ecological Indicators jo urnal homepage: www.elsevier.com/locate/ecolind Genetically modified whole-cell bioreporters for environmental assessment a b a,b a,∗ Tingting Xu , Dan M. Close , Gary S. Sayler , Steven Ripp a The University of Tennessee Center for Environmental Biotechnology, 676 Dabney Hall, Knoxville, TN 37996, USA b The Joint Institute for Biological Sciences, Oak Ridge National Laboratory, PO Box 2008, MS6342 Oak Ridge, TN 37831, USA a r t i c l e i n f o a b s t r a c t Article history: Living whole-cell bioreporters serve as environmental biosentinels that survey their ecosystems for Received 30 September 2011 harmful pollutants and chemical toxicants, and in the process act as human and other higher animal Received in revised form 16 January 2012 proxies to pre-alert for unfavorable, damaging, or toxic conditions. Endowed with bioluminescent, flu- Accepted 22 January 2012 orescent, or colorimetric signaling elements, bioreporters can provide a fast, easily measured link to chemical contaminant presence, bioavailability, and toxicity relative to a living system. Though well Keywords: tested in the confines of the laboratory, real-world applications of bioreporters are limited. In this review, Bioluminescence we will consider bioreporter technologies that have evolved from the laboratory towards true environ- Bioremediation Bioreporter mental applications, and discuss their merits as well as crucial advancements that still require adoption Ecotoxicology for more widespread utilization. Although the vast majority of environmental monitoring strategies rely Fluorescence upon bioreporters constructed from bacteria, we will also examine environmental biosensing through the use of less conventional eukaryotic-based bioreporters, whose chemical signaling capacity facilitates a more human-relevant link to toxicity and health-related consequences.

  • ARCHIVES of ENVIRONMENTAL PROTECTION Vol

    ARCHIVES OF ENVIRONMENTAL PROTECTION vol. 40 no. 4 pp. 113 - 123 2014 PL ISSN 2083-4772 DOI: 10.2478/aep-2014-0043 © Copyright by Polish Academy of Sciences and Institute of Environmental Engineering of the Polish Academy of Sciences, Zabrze, Poland 2014 ENVIRONMENTAL APPLICATION OF REPORTER-GENES BASED BIOSENSORS FOR CHEMICAL CONTAMINATION SCREENING MARZENA MATEJCZYK*, STANISŁAW JÓZEF ROSOCHACKI Bialystok University of Technology, Faculty of Civil and Environmental Engineering, Division of Sanitary Biology and Biotechnology, ul. Wiejska 45 E, 15-351 Bialystok, Poland * Corresponding author’s e-mail: [email protected] Keywords: Reporter genes, biosensors, chemical contamination. Abstract: The paper presents results of research concerning possibilities of applications of reporter-genes based microorganisms, including the selective presentation of defects and advantages of different new scientifi c achievements of methodical solutions in genetic system constructions of biosensing elements for environmental research. The most robust and popular genetic fusion and new trends in reporter genes technology – such as LacZ (β-galactosidase), xylE (catechol 2,3-dioxygenase), gfp (green fl uorescent proteins) and its mutated forms, lux (prokaryotic luciferase), luc (eukaryotic luciferase), phoA (alkaline phosphatase), gusA and gurA (β-glucuronidase), antibiotics and heavy metals resistance are described. Reporter-genes based biosensors with use of genetically modifi ed bacteria and yeast successfully work for genotoxicity, bioavailability and oxidative stress assessment for detection and monitoring of toxic compounds in drinking water and different environmental samples, surface water, soil, sediments. INTRODUCTION Nowadays, reporter genes technology is very useful and famous for widely application in different fi elds of sciences such as biology, ecology, medicine and environmental biotechnology [50].
  • In Vivo Analysis of Protein–Protein Interactions with Bioluminescence Resonance Energy Transfer (BRET): Progress and Prospects

    In Vivo Analysis of Protein–Protein Interactions with Bioluminescence Resonance Energy Transfer (BRET): Progress and Prospects

    International Journal of Molecular Sciences Review In Vivo Analysis of Protein–Protein Interactions with Bioluminescence Resonance Energy Transfer (BRET): Progress and Prospects Sihuai Sun †, Xiaobing Yang †, Yao Wang and Xihui Shen * State Key Laboratory of Crop Stress Biology for Arid Areas and College of Life Sciences, Northwest A&F University, Yangling 712100, Shaanxi, China; [email protected] (S.S.); [email protected] (X.Y.); [email protected] (Y.W.) * Correspondence: [email protected]; Tel.: +86-29-8709-2087 † These authors contributed equally to this work. Academic Editors: Christo Z. Christov and Tatyana Karabencheva-Christova Received: 15 August 2016; Accepted: 29 September 2016; Published: 11 October 2016 Abstract: Proteins are the elementary machinery of life, and their functions are carried out mostly by molecular interactions. Among those interactions, protein–protein interactions (PPIs) are the most important as they participate in or mediate all essential biological processes. However, many common methods for PPI investigations are slightly unreliable and suffer from various limitations, especially in the studies of dynamic PPIs. To solve this problem, a method called Bioluminescence Resonance Energy Transfer (BRET) was developed about seventeen years ago. Since then, BRET has evolved into a whole class of methods that can be used to survey virtually any kinds of PPIs. Compared to many traditional methods, BRET is highly sensitive, reliable, easy to perform, and relatively inexpensive. However, most importantly, it can be done in vivo and allows the real-time monitoring of dynamic PPIs with the easily detectable light signal, which is extremely valuable for the PPI functional research.
  • Escherichia Coli Ribose Binding Protein Based Bioreporters Revisited

    Escherichia Coli Ribose Binding Protein Based Bioreporters Revisited

    OPEN Escherichia coli ribose binding protein SUBJECT AREAS: based bioreporters revisited APPLIED MICROBIOLOGY Artur Reimer1, Sharon Yagur-Kroll2, Shimshon Belkin2, Shantanu Roy1 & Jan Roelof van der Meer1 ASSAY SYSTEMS 1Department of Fundamental Microbiology, University of Lausanne, Baˆtiment Biophore, Quartier UNIL-Sorge 1015 Lausanne, 2 Received Switzerland, Department of Plant and Environmental Sciences, The Alexander Silberman Institute of Life Sciences, The Hebrew 8 April 2014 University of Jerusalem, Jerusalem 91904, Israel. Accepted 17 June 2014 Bioreporter bacteria, i.e., strains engineered to respond to chemical exposure by production of reporter proteins, have attracted wide interest because of their potential to offer cheap and simple alternative Published analytics for specified compounds or conditions. Bioreporter construction has mostly exploited the natural 9 July 2014 variation of sensory proteins, but it has been proposed that computational design of new substrate binding properties could lead to completely novel detection specificities at very low affinities. Here we reconstruct a bioreporter system based on the native Escherichia coli ribose binding protein RbsB and one of its computationally designed variants, reported to be capable of binding 2,4,6-trinitrotoluene (TNT). Our Correspondence and results show in vivo reporter induction at 50 nM ribose, and a 125 nM affinity constant for in vitro ribose requests for materials binding to RbsB. In contrast, the purified published TNT-binding variant did not bind TNT nor did TNT should be addressed to cause induction of the E. coli reporter system. J.R.M. (janroelof. [email protected]) onstruction of bioreporter bacteria typically starts with identifying a sensory protein that controls express- ion of a target gene promoter in dependence on one or more chemical inducers1–8.
  • Highly Sensitive, Highly Specific Whole-Cell Bioreporters for the Detection of Chromate in Environmental Samples

    Highly Sensitive, Highly Specific Whole-Cell Bioreporters for the Detection of Chromate in Environmental Samples

    Highly Sensitive, Highly Specific Whole-Cell Bioreporters for the Detection of Chromate in Environmental Samples Rita Branco1,2, Armando Cristo´ va˜o3,4, Paula V. Morais1,4* 1 IMAR, 3004-517 Coimbra, Portugal, 2 Escola Universita´ria Vasco da Gama, Mosteiro de S. Jorge de Milre´u, Estrada da Conraria, Castelo Viegas – Coimbra, Portugal, 3 Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal, 4 Department of Life Sciences, FCTUC, University of Coimbra, Coimbra, Portugal Abstract Microbial bioreporters offer excellent potentialities for the detection of the bioavailable portion of pollutants in contaminated environments, which currently cannot be easily measured. This paper describes the construction and evaluation of two microbial bioreporters designed to detect the bioavailable chromate in contaminated water samples. The developed bioreporters are based on the expression of gfp under the control of the chr promoter and the chrB regulator gene of TnOtChr determinant from Ochrobactrum tritici 5bvl1. pCHRGFP1 Escherichia coli reporter proved to be specific and sensitive, with minimum detectable concentration of 100 nM chromate and did not react with other heavy metals or chemical compounds analysed. In order to have a bioreporter able to be used under different environmental toxics, O. tritici type strain was also engineered to fluoresce in the presence of micromolar levels of chromate and showed to be as specific as the first reporter. Their applicability on environmental samples (spiked Portuguese river water) was also demonstrated using either freshly grown or cryo-preserved cells, a treatment which constitutes an operational advantage. These reporter strains can provide on-demand usability in the field and in a near future may become a powerful tool in identification of chromate-contaminated sites.
  • Bioreporter Bacteria-Based Test Systems

    Bioreporter Bacteria-Based Test Systems

    3K''LVVHUWDWLRQ ,PSURYHPHQWRIELRUHSRUWHUEDFWHULDEDVHGWHVWV\VWHPVIRU WKHDQDO\VLVRIDUVHQLFLQGULQNLQJZDWHUDQGWKHUKL]RVSKHUH $QNH.XSSDUGW ,661 Improvement of bioreporter bacteria-based test systems for the analysis of arsenic in drinking water and the rhizosphere Von der Fakultät für Biowissenschaften, Pharmazie und Psychologie der Universität Leipzig genehmigte D I S S E R T A T I O N zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) vorgelegt von Diplom Geoökologin Anke Kuppardt, geb. Wackwitz geboren am 24.12.1979 in Burgstädt Dekan: Prof. Dr. Matthias Müller Gutachter: Prof. Dr. Hauke Harms Prof. Dr. Jan Roelof van der Meer Tag der Verteidigung: 05. Februar 2010 To my dear husband and my dear son for supporting and distracting me Abstract Contamination of drinking water with arsenic can be measured in laboratories with atom absorption spectrometry (AAS), mass spectrometry with inductive coupled plasma (ICP-MS) or atom fluorescence spectrometry (AFS) at the relevant concentrations below 50 µg/L. Field test kits which easily and reliably measure arsenic concentrations are not yet available. Test systems on the basis of bioreporter bacteria offer an alternative. Based on the natural resistance mechanism of bacteria against arsenic compounds toxic for humans, bioreporter bacteria can be constructed that display arsenic concentrations with light emission (luminescence or fluorescence) or colour reactions. This is achieved by coupling the gene for the ArsR-protein and arsenic regulated promoters with suitable reporter genes. The resulting bioreporter bacteria report bioavailable arsenic in a dose dependent manner at the toxicologically relevant level of 2 to 80 µg/L and are therewith suitable both for the guideline levels of the WHO of 10 µg/L and for the national standards in South East Asia of 50 µg/L.
  • Bioluminescent Bioreporter Pad Biosensor for Monitoring Water Toxicity

    Bioluminescent Bioreporter Pad Biosensor for Monitoring Water Toxicity

    See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/284691524 Bioluminescent bioreporter pad biosensor for monitoring water toxicity Article in Talanta · November 2015 Impact Factor: 3.55 · DOI: 10.1016/j.talanta.2015.11.067 READS 74 3 authors: Tim Axelrod Evgeni Eltzov Ben-Gurion University of the Negev Ben-Gurion University of the Negev 1 PUBLICATION 0 CITATIONS 29 PUBLICATIONS 274 CITATIONS SEE PROFILE SEE PROFILE Robert S Marks Ben-Gurion University of the Negev 185 PUBLICATIONS 2,648 CITATIONS SEE PROFILE All in-text references underlined in blue are linked to publications on ResearchGate, Available from: Evgeni Eltzov letting you access and read them immediately. Retrieved on: 22 May 2016 Talanta 149 (2016) 290–297 Contents lists available at ScienceDirect Talanta journal homepage: www.elsevier.com/locate/talanta Bioluminescent bioreporter pad biosensor for monitoring water toxicity Tim Axelrod a, Evgeni Eltzov a,b, Robert S. Marks a,b,c,d,n a Department of Biotechnology Engineering, Faculty of Engineering Science, Ben-Gurion University of the Negev, Beer-Sheva, Israel b School of Material Science and Engineering, Nanyang Technology University, Nanyang Avenue, 639798 Singapore c National Institute of Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva, Israel d The Ilse Katz Center for Meso and Nanoscale Science and Technology, Ben-Gurion University of the Negev, Beer-Sheva, Israel article info abstract Article history: Toxicants in water sources are of concern. We developed a tool that is affordable and easy-to-use for Received 1 October 2015 monitoring toxicity in water.