WO2006018049A1 - Système d’expression presenté en surface - Google Patents

Système d’expression presenté en surface Download PDF

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Publication number
WO2006018049A1
WO2006018049A1 PCT/EP2005/001707 EP2005001707W WO2006018049A1 WO 2006018049 A1 WO2006018049 A1 WO 2006018049A1 EP 2005001707 W EP2005001707 W EP 2005001707W WO 2006018049 A1 WO2006018049 A1 WO 2006018049A1
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Prior art keywords
stress
host cell
drug
cells
assayable
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PCT/EP2005/001707
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English (en)
Inventor
Wim De Coen
Yves Guisez
Marleen Maras
Johan Robbens
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Aic
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Priority claimed from PCT/EP2004/009265 external-priority patent/WO2005017198A2/fr
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Publication of WO2006018049A1 publication Critical patent/WO2006018049A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C12N15/625—DNA sequences coding for fusion proteins containing a sequence coding for a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/035—Fusion polypeptide containing a localisation/targetting motif containing a signal for targeting to the external surface of a cell, e.g. to the outer membrane of Gram negative bacteria, GPI- anchored eukaryote proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]

Definitions

  • This invention relates to the field of genetic engineering, molecular biology, (eco)toxicology and pharmacology.
  • the present invention provides a biological surface display expression system for detecting stress-inducing compounds.
  • the present invention also relates to methods for identifying and characterizing stress-inducing compounds and diagnostic kits for performing such methods.
  • living representative indicator organisms are used as biological monitors.
  • the simplest and most convenient of these systems utilize unicellular microorganisms, since they are most easily maintained and manipulated.
  • bacterial assays have been developed.
  • Such assays are colorimetric, luminescent or fluorescent assays comprising the expression of reporter genes encoding an assayable product, for instance a luminescent protein, under the control of inducible stress-responsive promoters in bacteria. For instance, Sagi et al.
  • the signals generated by the stress-inducing compounds can be distorted by interfering compounds present in the environmental matrix or the extract thereof.
  • the interfering compounds can interfere directly during signal measurement.
  • Shi et al. 2001 (Enzyme and microbial technology, 28 (1), p25-34) relates to the expression of GFP on the external cell surface of E. coli, by using a construction of a tripartite fusion protein, consisting of an OmpA domain and the mature GFP sequence, fused downstream of the first nine N-terminal amino acids of the mature outer membrane lipoproteine Lpp.
  • the Lpp-OmpA-GFP fusion protein is put under the control of an inducible promoter, in particular a lac promoter.
  • the constructed vector was introduced in E. coli, and GFP was targeted to the outer membrane of E. coli.
  • the reported method is not suitable for detecting stress-inducing (toxic and or xenobiotic) compounds.
  • Yet another object of the present invention consists of providing a test system and a method for characterizing the effects of an environmental matrix or an extract thereof on cells.
  • the present invention also aims to provide a test system and a method for characterizing the interaction of different co-cultured host cell types either in the presence or the absence of (stress-inducing) compounds.
  • the invention aims to provide a method that allows circumventing the commonly encountered problems with testing environmental samples or extracts such as e.g. color and turbidity.
  • the present invention provides a highly sensitive biological reporter system for detecting and characterizing toxicity levels of compounds as well as their toxicological mode of action.
  • the invention relates to a biological reporter system displayed at the surface of a host cell.
  • the present invention relates to a construct consisting of a chimeric gene encoding an assayable product and expressible in a host cell preceded by a toxicologically- inducible promoter, said chimeric gene comprising
  • construct refers to a chimeric gene as defined herein that is fused to and of which the expression is under the control of a toxicologically-inducible promoter as defined herein.
  • RNA damage all compounds which are foreign to the organism (cell) under study, including but not limited to, chemicals, antibiotics, environmental pollutants, heavy metals, as well as agents producing oxidative damage, DNA damage, RNA damage, or anaerobiosis.
  • toxicologically inducible promoter refers to a promoter of a gene which is activated in a cell due to the active presence of a certain compound.
  • the term “toxicologically-inducible promoter” therefore does not encompass promoters of genes that are activated in a cell as a result of the lower concentration or even the absence of a certain compound or nutrient.
  • the toxicologically-inducible promoter preferably shows a progressive induction pattern.
  • the term "showing a progressive-induction pattern”, as used herein refers to a toxicologically-inducible promoter which shows an induction level that is gradually increasing after induction. This term does not include promoters which show a maximal induction level at a single time-point, i.e. at the moment of induction. In addition, such promoter is further preferably rapidly induced and shows a limited background activity.
  • the toxicologically-inducible promoter shows a time-dependent progressive induction pattern.
  • time-dependent progressive induction pattern refers to the fact that the promoter does not reach its maximal induction level at a single time-point, i.e. when induced, but that the promoter shows a gradual increase of its induction level in function of exposure time up to a maximal induction level.
  • the toxicologically-inducible promoter shows a dose- dependent progressive induction pattern.
  • dose-dependent progressive induction pattern it is meant that the promoter generates a proportional response of transcription over a high dynamic range of concentrations of toxic compounds that are tested.
  • the promoter shows an induction level that is a function of the concentration of toxic compound and that is gradually increasing with increasing concentrations of toxic compound.
  • the present promoter is able to show a progressive increase in induction in function of concentration of toxic compounds over a concentration gradient that spans at least one order of magnitude and preferably at least two, at least three, at least four and preferably at least five orders of magnitude.
  • the toxicologically inducible promoter shows a time dependent as well as a dose dependent progressive induction pattern.
  • rapidly induced refers to the ability to provide a detectable transcription signal within at least 6 hours after induction, and preferably within at least 4 hours after induction, and preferably within at least 2 hours after induction, and more preferably within at least 1.5 hour after induction and most preferably within at least 1 hour after induction.
  • a limited background activity refers to a promoter activity that is as low as possible.
  • an inducible promoter according to the invention promotes gene expression under non-induced conditions at a level that is lower than 10%, and preferably lower than 8%, and more preferably lower than 5%, and even more preferred lower than 2.5 %, and most preferred lower than 1% of the maximal expression level promoted by the promoter under maximal toxic stress conditions.
  • the biological reporter system according to the invention is displayed at the surface of the detector-organism, being a prokaryotic cell.
  • the DNA sequence which encodes an assayable product, is preceded by a segment of a transmembrane sequence for anchoring and exposing said assayable product in the cell membrane of a host cell towards the extracellular medium.
  • the DNA segment encoding a transmembrane amino acid sequence is preceded by a targeting DNA sequence for targeting said assayable product to said host cell membrane.
  • the assayable product of the reporter gene will be targeted on the cell surface of the host cell and suitably anchored in the host cell membrane, in order to allow a correct exposure of the reporter gene product to the extracellular space.
  • the stress-inducible reporter system allows on-line and immediate detection of the molecular response of the reporter protein without prior lysis or additional manipulation of the cell population.
  • membrane display is used in combination with a toxicologically-inducible promoter.
  • a toxicologically-inducible promoter e.g. fluorescence
  • a toxicologically-inducible promoter as defined herein for controlling the expression of above-described chimeric gene provides several important advantages. It enables to identify and detect toxic compounds in a sample. It further allows performing toxicological assays having both a quantitative and qualitative character. These two characteristics are the main characteristics for a desirable toxicological characterization of a molecule.
  • the present invention further relates to recombinant vectors carrying a construct according to the present invention, and to a host cell transformed with such vector.
  • the present invention relates to methods for identifying and characterizing a stress-inducing compound in a sample (e.g. environmental pollutants), for identifying and characterizing the toxicity of a drug, for identifying and characterizing an antitoxin to a stress-inducing compound or drug or for identifying and characterizing a stress- inducing compound or drug having a decreased toxicity.
  • a stress-inducing compound in a sample e.g. environmental pollutants
  • the present invention also relates to a test system and methods for identifying and characterizing the effects of an environmental matrix or an extract thereof on host cells.
  • the present invention also provides a test system and methods for identifying and characterizing interactions between different co-cultured host cell types either in the presence or the absence of (stress-inducing) compounds.
  • the methods according to the present invention comprise direct and/or indirect detection of the assayable product displayed at the surface of the host cell.
  • Direct detection methods may comprise colorimetric, fluorimetric, luminescence or flow cytometric detection techniques.
  • Indirect detection methods are preferably based on immunolabelling such as flow cytometric techniques, immunoassays, Western blots etc...
  • any other or additional physico-chemical measurement technique may be used for detection of the assayable product displayed at the surface of a host cell, such as but not limited to e.g. measurement techniques based on protein-protein-interactions, ligand-protein and receptor-ligand interactions, electrode-based interaction, etc..
  • the present invention also provides diagnostic kits for performing the methods according to the present invention.
  • the methods and kits according to the invention are particularly suitable for the identification and evaluation of stress-inducing compounds in general, and of mutagens in particular, which are present in the environment.
  • the methods and kits are also particularly suitable for identifying the toxicity of drugs, and for use in drug design.
  • the present invention relates to a novel technology, constructs, methods and diagnostic kits for detecting and characterizing stress responses in cells caused by stress-inducing compounds or drugs, and to identify and characterize compounds or drugs having a stress- inducing activity.
  • the present invention further relates to a reporter system, constructs, methods and diagnostic kits for detecting and determining the mode of action of stress- inducing compounds or drugs.
  • the present invention also relates to a novel technology, methods and diagnostic kits particularly suitable for identifying and characterizing interactions of different cell cultures when co-cultured, either in the presence or the absence of (stress- inducing) compounds.
  • a particular class of stress-inducing compounds includes the genotoxic chemicals or mutagens.
  • “Genotoxic chemicals” or “mutagens”, as used herein refer to substances or agents that cause DNA damage in a cell. Such damage can potentially lead to the formation of a malignant tumor, but DNA damage does not lead inevitably to the creation of cancerous cells.
  • alteration of DNA can take place through a variety of mechanisms, which are known to be chemical-specific.
  • nucleotides can be alkylated, oxidated, deaminated or hydroxylated.
  • Bulky products can be covalently added to nucleotides to destabilize DNA- basepairing or base analogs can be incorporated.
  • Other mutagens intercalate between the DNA strands and provoke insertion- or deletion mutations.
  • drug refers to pharmaceutical compositions or medicaments.
  • the present invention relates to a chimeric gene encoding an assayable product and expressible in a host cell which is fused to a toxicologically-inducible promoter.
  • the toxicologically-inducible promoters used according to the present invention preferably are informative on the type of toxicological damage that is occurring in the cell.
  • Toxicological informative means that mechanisms of the toxicological mode of action in a cell can be elucidated, such as 1) information on which type of molecule (lipid, protein, nucleic acid, carbohydrate, vitamin, hormone... or combinations hereof) or pathway that is affected by the chemical; 2) information on a repair mechanism that is initiated or induced inside the cell; 3) information on the type of elimination, excretion or biotransformation pathway that is affected or a combination of these 3 steps.
  • the type of promoter that is used for such purpose depends on the types of molecules or types of damage that are investigated.
  • a toxicologically-inducible promoter is used to control the expression of above-described chimeric gene.
  • a "toxicologically-inducible promoter" as used herein refers to the promoter of a gene responsive to a stress condition such as but not limited to heat stress, cold stress, redox stress, DNA stress, RNA stress, lipid stress, protein stress, energy stress, osmotic stress, pH stress or membrane stress.
  • a stress condition such as but not limited to heat stress, cold stress, redox stress, DNA stress, RNA stress, lipid stress, protein stress, energy stress, osmotic stress, pH stress or membrane stress.
  • stress promoter induction refers to conditions, which increase or decrease the level of expression of assayable gene product.
  • heat stress refers to conditions, which disrupt cellular metabolism in a cell, and may be induced by heat stress inducing factors such as heat.
  • cold stress refers to conditions, which disrupt cellular metabolism in a cell, and may be induced by cold stress inducing factors such as cold.
  • redox stress refers to conditions which vary from the normal reduction/oxidation potential (“redox”) state of the cell. Redox stress includes increased levels of superoxides, increased levels of peroxides, both hydrogen peroxide and organic peroxides, decreased levels of glutathione and any other conditions which alter the redox potential of the cell, such as exposure to strong reducing agents.
  • DNA stress refers to alterations to deoxyribonucleic acid or to precursor nucleotides.
  • DNA stress includes, but is not limited to, DNA strand breaks, DNA strand crosslinking, ionizing stress, exposure to DNA intercalating agents, both increased and decreased superhelicity, oxidative DNA damage, DNA alkylation, oxidation of nucleotide triphosphates and alkylation of nucleotide triphosphates.
  • RNA stress refers to alterations to ribonucleic acid or to precursor nucleotides.
  • RNA stress includes, but is not limited to, RNA strand breaks, ionizing stress, exposure to RNA intercalating agents, oxidative RNA damage, RNA alkylation, oxidation of nucleotide triphosphates and alkylation of nucleotide triphosphates.
  • the term also includes inhibition of RNA synthesis.
  • lipid stress refers to alterations to lipids or individual fatty acids, as well as perturbations of intracellular transport of lipids.
  • the term includes, but is not limited to, denaturation of lipids, both oxygen dependent and oxygen independent oxidation lipids, alkylation of lipids, oxidation of individual fatty acids and lipid damage caused by exposure to heavy metals, such as cadmium.
  • Protein stress refers to alterations to proteins or individual amino acids, as well as perturbations of intracellular transport of proteins.
  • the term includes, but is not limited to, denaturation of proteins, misfolding of proteins, chelation of protein cofactors, cross-linking of proteins, both oxygen dependent and -independent oxidation of inter- and intra-chain bonds, such as disulfide bonds, alkylation of proteins, oxidation of individual amino acids and protein damage caused by exposure to heavy metals, such as cadmium.
  • energy stress encompasses conditions which affect ATP levels in the cell. Examples of energy stress are forced anaerobic metabolism in the presence of oxygen, perturbations of electron transport and exposure to uncoupling agents.
  • osmotic stress refers to conditions, which cause perturbations in the maintenance of the internal osmolarity of a cell at a relatively invariant level in face of fluctuations in the osmolarity of the environment.
  • pH stress refers to conditions, which cause perturbations in intracellular pH, i.e., which decrease intracellular pH below about 6.0 or increase intracellular pH above about 7.5. pH stress may be caused, for example, by exposure of the cell to ionophores or other cell membrane damaging components, or exposure to weak organic hydrophobic acids, such as phenolic acid. The term also includes cell membrane damage and deleterious changes in electromotive potential.
  • membrane stress refers to conditions which perturbations in the organisms' membrane(s).
  • Suitable stress-inducible promoters for use in prokaryotic cells may thus include but are not limited to promoters of genes responsive to heat stress, cold stress, redox stress, DNA stress, RNA stress, lipid stress, protein stress, energy, osmotic stress or pH stress.
  • promoter of genes of which the expression is altered upon other type of stress conditions can be used as stress- inducible promoters.
  • suitable toxicologically-inducible promoters may also include promoters of genes that are involved in cellular detoxification mechanisms, recombination, DNA and RNA repair mechanisms, SOS repair mechanisms, etc...
  • new toxicologically-inducible promoters that may be discovered and characterized may also be employed in the methods and kits of this invention.
  • promoter parameters to which a toxicologically-inducible promoter in accordance with the present invention needs to fit includes showing a time-dependent and/or a dose- dependent progressive induction pattern.
  • Promoters related to toxicological events are suitable in accordance with the present invention, since they control the expression of genes involved in protection mechanisms of a cell and preservation mechanisms of its vital functions depending on the level of damage or impact that is occurring.
  • promoters are desired for driving a chimeric gene according to the invention, that show a rapid induction after a short time frame.
  • a sufficient signal could be measured after less than 6 hours, and in particular after less than 4 hours, and even better after less than 2 hours, and even better less than 1 hour.
  • screening assays are usually performed in a semi high-throughput environment wherein toxicity of a sample or compound is preferably evaluated in the shortest possible time frame.
  • a toxicologically-inducible promoter used in accordance with the present invention preferably further shows a limited background activity.
  • Promoter leakage is unacceptable, considering the mentioned required dynamic dosage effect, but more important here is the necessity to detect all possible toxic effect, however without drawing wrong conclusions from 'false positives'.
  • promoter promotes gene expression under non-induced conditions at a level that is lower than 10%, and preferably lower than 5%, of the maximal expression level promoted by the promoter under maximal toxic stress conditions.
  • the present invention provides an essentially positive biological reporter system and method.
  • positive is meant that a positive (increased) signal, e.g. increased fluorescence, is detected in the presence of a toxic compound.
  • the biological reporter system is based on the following approach: a promoter of a progressive stress-inducible nature linked to a toxicologically relevant phenomenon is linked to a gene, i.e. a "reporter gene", which produces a measurable product.
  • This reporter gene has been inserted into a cell, which responds to stress by making the reporter gene product.
  • the reporter gene has been genetically designed for rapidly and easily providing an assayable reporter gene product activity at the surface of a host cell. Display and attachment at the surface of the detector-organism enables on line and easy detection of a reporter gene product without additional lysis or manipulation of the host cell. Readout of the results can be carried out rapidly and simply with the intact organism, without the necessity of disruption of the cell or extraction of the polypeptide or enzyme to be measured.
  • the assay can be performed easily in the laboratory or in the field, by personnel with minimal training.
  • a number of suitable promoters may be used.
  • Good examples of toxicologically-inducible promoters for stress-related phenomena are given in WO 94/13831 , which is incorporated herein by reference.
  • Non-limitative examples of suitable toxicologically-inducible promoters are further listed in Table 1 (example 1).
  • Preferred examples of toxicologically-inducible promoters useful in accordance with the present invention involve but are not limited to a promoter of one of the following genes ada, ada-alkA, ahp, aniG, ars, cad, cyo, cyd, clpP, clpB, dnaK, dinA, dinB, dinD, dinF, fepB-entC, fabA, frdA, glpQ, groE, groES, groEL, grpE, gsh, gyr, htpE, htpG, htpl,m MpK 1 MpO, MpX, htpN, hag, katF, katG, Ion, lexA, lysU, leu-500, micF, eto, mutT, merR, mer, nfo, narG, osmY,
  • the present invention relates to a construct consisting of a chimeric gene preceded by a promoter of a gene responsive to heat stress such as but not limited to clpB, dnaK, hscA, hslS, hslT, hslS, hslT, hslU, hslV, MgA, MpG, MpX, MrA.
  • the present invention relates to a construct consisting of a chimeric gene preceded by a promoter of a gene responsive to redox stress such as but not limited to glpQ, speF, soxRS.
  • the present invention relates to a construct consisting of a chimeric gene preceded by a promoter of a gene responsive to protein stress such as but not limited to clpB, hrsA, hslV, lysU, pepT.
  • the present invention relates to a construct consisting of a chimeric gene preceded by a promoter of a gene responsive to DNA stress such as but not limited to recA, recF, recG, recN, phrB, polA, polB.
  • the present invention relates to a construct consisting of a chimeric gene preceded by a promoter of a gene responsive to RNA stress such as but not limited to recJ, rpoA, rpoB, rpoC, rpoD, rpoH, rpoS.
  • the present invention relates to a construct consisting of a chimeric gene preceded by a promoter of a gene responsive to lipid stress such as but not limited to cfa, fadB.
  • the present invention relates to a construct consisting of a chimeric gene preceded by a promoter of a gene responsive to energy stress such as but not limited to fdhF, fdnG, fdnH, fdnl, glpA, glpB, glpC, glpD, glpG.
  • the present invention relates to a construct consisting of a chimeric gene preceded by a promoter of a gene responsive to osmotic stress such as but not limited to mdoB, mdoH, osmB, osmC, osmE, osmY, otsA, otsB.
  • the present invention relates to a construct consisting of a chimeric gene preceded by a promoter of a gene responsive to pH stress such as but not limited to polA, speF, rpoS.
  • the present invention relates to a construct consisting of a chimeric gene preceded by a promoter of a gene responsive to membrane stress such as but not limited to cfa.
  • the toxicologically-induced promoter for use in the present construct is selected from the group comprising a recA, katG, zwf, soi28, osmY, micF, clpB, merR, ada, dinD, nfo, uspA, umuDC or soxRS promoter.
  • targeting DNA sequence is intended to indicate a sequence encoding a polypeptide capable of targeting the fusion polypeptide, encoded by the chimeric gene, to the host cell membrane.
  • host cell membrane may refer to different cell structures depending on the type of host cell applied according to the invention. In an example, this term refers to the inner membrane of G " bacteria or to the cell membrane of G + bacteria.
  • Lpp E. coli lipoprotein
  • the E. coli Lpp targeting sequence includes the signal sequence and the first 9 amino acids of the mature protein.
  • Lpp Lpp
  • Other secreted proteins from which targeting sequences may be derived include TraT, OsmB, KIpB, phoA (phosphataseA), MBP (maltose binding protein), bla ( ⁇ -lactamase), dsbA (disulphide binding protein), npr (phosphoryl acceptor) or heat labile toxin STII.
  • Lipoprotein 1 from Pseudomonas aeruginosa or the PA1 and PCN proteins from Haemophilus influenza as well as the 17 kDa lipoprotein from Rickettsia rickettsij and the H.8 protein from Neisseria gonorrhea and the like may be used in a construct according to the present invention.
  • transmembrane amino acid sequence is intended to denote an amino acid sequence capable of transporting a polypeptide through the membrane of a host cell and to assure an efficient membrane anchoring and correct exposure of the polypeptide to the external surface of the host cell.
  • Transmembrane proteins serve a different function from that of targeting sequences and generally include amino acid sequences longer than the polypeptide sequences effective in targeting proteins to the host cell membrane.
  • DNA sequences encoding a transmembrane amino acid sequence are well known and have been identified in several prokaryotic organisms, including G+ bacteria and G- bacteria. Non-limitative examples of genes comprising transmembrane amino acid sequences suitable for use in a construct according to the present invention are listed in Table 2.
  • the DNA sequence encoding an assayable product is any heterologous or homologous protein or enzyme that can be expressed in a prokaryotic cell.
  • the DNA sequence encoding an assayable product is a reporter gene. This DNA sequence is positioned downstream from the DNA segment encoding the transmembrane sequence.
  • reporter gene refers to nucleic acid sequences encoding assayable proteins. The choice of reporter genes to be used is essentially limitless, as long as a DNA sequence encoding the assayable product has been characterized; and the product of the gene can be detected. Sufficient characterization includes knowledge of the entire coding sequence and availability of a cDNA molecule.
  • the DNA sequence encoding an assayable product may include a DNA sequence encoding an enzyme or protein selected from the group comprising ATPases 5 , Multi-enzyme Complexes, Cytochromes, Multifunctional Enzymes, DNA Restriction-Modification Enzymes, Mutases, Deaminases, Nucleases, Decarboxylases, Oxidases, Dehydrogenases, Oxioreductases, Desaturases, Peptidases, Dioxygenases, Permeases, Elastases, Peroxidases, Endopeptidases, Phosphatases, Flavoproteins, Phosphorylases, Flippases, Proteolytic Enzymes, Hydrogenases, Recombinases, Hydrolases, Reductases, Hydroxylases, Regulatory Enzymes, Integrases, Ribonucleases, Isomerases, Sulfatases, Kinases, Synthases, Ligases, Synthetases, Lipoxygen
  • the assayable product is, chloramphenicol acetyl transferase (encoded by the cat gene), galactose kinase (encoded by the galK gene), /?-glucosidase (encoded by the gus gene), glutathione transferase or luciferase (encoded by the lux gene),or green fluorescent protein (encoded by the GFP gene) or mutants thereof, Dsred, . ⁇ galactosidase, ⁇ -glucuronidase, ⁇ -lactamase, etc....
  • the GFP gene is employed, and even more preferably a mutated version of the GFP gene, for instance GFPmut2 is used.
  • any other gene encoding an assayable protein including newly identified genes, may be used in accordance with the present invention.
  • Toxicologically-lnducible promoters used in the present invention must be operatively linked to the chimeric gene.
  • operative linkage refers to the positioning of the promoter relative to the chimeric gene encoding the assayable product such that transcription of the gene is regulated by the promoter. Such positioning is well known in the art and involves positioning the promoter upstream (5 1 ) of the gene so that no transcription termination signals are present between the promoter and the gene.
  • a promoter sequence Operatively linked' to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the promoter sequence.
  • assayable product refers to a product, e.g. protein, that is displayed at the surface of a host cell and that can be detected by any type detection method, including direct and/or indirect detection methods.
  • the assayable gene product can be measured both qualitatively and quantitatively by means of any type of detection method including but not limited to any physico-chemical measurement technique such as fluorescence, absorbance, conductivity, magnetic resonance, measurement techniques based on protein-protein, ligand-protein and receptor-protein interactions, electrode-based interaction, etc; any immunolabelling technique in its broadest context, or any other specific detection technique.
  • the surface display allows assessment by using immunolabelling techniques.
  • immunolabelling technique as used herein is meant to refer to various detection methods that use immunoglobulins to detect specific epitopes.
  • immunolabelling technique may comprise but are not limited to ELISA, immunostaining, immunohistochemistry, enzyme immunoassays, Western Blotting, Flow Cytometry, Nephelometry, immunosensors, etc...
  • the invention relates to a construct, which is expressible in a prokaryotic host cell selected from the group comprising G+ bacteria and G- bacteria.
  • the invention relates to a recombinant vector carrying a construct according to any of the embodiments of the invention.
  • the invention in another further embodiment, relates to a host cell transformed with a vector according to the invention.
  • a recombinant vector carrying a chimeric gene according to any of embodiments of the invention can be introduced into a host cell using standard recombinant DNA techniques that are well known in the art.
  • the present invention relates to a prokaryotic host cell transformed with a vector according to the invention.
  • transformation refers to the acquisition of new genes in a cell after the incorporation of nucleic acid.
  • Said host cell may be a prokaryotic cell selected from the group comprising G+ bacteria and G- bacteria. Any of a wide variety of G- bacteria may be useful in practicing the invention. Such G- bacteria may include E. coli, Salmonella, Klebsiella, Erwinia, and the like. Any of a wide variety of G+ bacteria may equally be useful in practicing the invention. Such G+ bacteria may include Staphylococcus sp., Bacillus sp., and the like.
  • the present invention further relates to methods and diagnostic kits for identifying and characterizing stress-inducing compounds.
  • Such methods and kits comprise at least one host cell which has been transformed with the above-described recombinant vector. Identification and characterization of stress-inducing compounds is achieved by detecting the assayable product displayed at the surface of such host cell.
  • the present invention relates to a method for identifying and characterizing a stress-inducing compound in a sample comprising the steps of: - separately culturing one or more of the above-described host cells,
  • the present invention further relates to a method for detecting and determining the mode of action of a stress-inducing compound in a sample comprising the steps of:
  • the above described method is for instance particularly suitable for monitoring samples for the presence of stress-inducing compounds in general or of genotoxic or mutagenic compounds in particular.
  • stress-inducing compounds can be distinguished using a system according to the invention. Potential uses include monitoring of air, soil, water and food quality, agrochemical and drug design, manufacturing and fermentation process control, process monitoring and toxicity screening. These applications may benefit many industries including chemical, beverage, food and flavor, cosmetics, agricultural, environmental, regulatory and health care industries.
  • the present invention relates to a method assay wherein the sample to be analyzed is selected from the group comprising an aqueous solution, water, soil, sediment, sludge, food, beverage or pesticides.
  • each employed host cell harbors only one particular stress promoter-chimeric gene fusion.
  • the specific type and mode of action caused by the stress-inducing or mutagen compound can unambiguously be identified.
  • the copy number of each stress promoter- chimeric gene fusion utilized in the methods and kits of this invention is equal.
  • the method according to this invention comprises the first step of separately culturing each of the individual hosts, according to methods well known in the art. For instance, bacterial host cells are grown so that they are in log or stationary phase. Growth may be in minimal media, with or without antibiotics, such as depending on the strain of bacteria used. Growth of the hosts is followed by measuring cell density via absorbance of the culture at 600 nm (OD 600 ). Following this initial growth, a sample wherein a stress-inducing compound, or in particular a mutagen, may be present, is added to one fraction of each culture. The other fraction of each culture is not exposed to the solution or extract, and is used as both a control to measure the effect of the compound on the overall growth of the cells and for a baseline measurement of assayable gene product.
  • a stress-inducing compound or in particular a mutagen
  • the OD 600 of the cultures just prior to exposure to the compound is recorded. All of the cultures, both control and exposed, are then allowed to incubate at normal growth temperature for a period of time ranging from 5 minutes to 24 hours. More preferably, exposure to the stress-inducing or test compound is for about 30 minutes to 4 hours. After this additional incubation, both the exposed and control cultures are used to determine comparative growth by measuring OD 600 .
  • mutagenized cells can have a growth advantage over non- mutagenized cells in particular cases wherein an essential gene is mutagenized in said cells.
  • the present invention relates to a method for identifying and characterizing the toxicity of a stress-inducing compound or drug comprising the steps of: separately culturing one or more of the above-described host cells, - incubating said one or more cultures of said cells with said stress-inducing compound or drug at one or more concentrations, - detecting an assayable product displayed at the surface of said host cell in each of said cultures.
  • the present invention provides a method for determining and characterizing the toxicity of a stress-inducing compound or drug in terms of the type of stress it causes within the cell. Such methods are particularly suitable for determining stress-inducing effects of drugs. In the frame of registration procedures of drugs for human or animal use, such studies are particularly relevant.
  • kits and methods of this invention can also be utilized to determine the potential toxicity of combinations of known and unknown compounds in an identical manner to that described above.
  • the invention provides a method for identifying an antitoxin to a compound determined to induce stress by the methods of this invention.
  • the present invention relates to a method for identifying and characterizing an antitoxin to a stress-inducing compound or drug comprising the steps of:
  • an antitoxin to said known stress-inducing compound, said antitoxin being also suitable to act as an antitoxin for said stress-inducing compound or drug.
  • a stress promoter induction/suppression profile can be generated for a known or unknown stress-inducing compound or drug, that profile is compared to profiles of known substances in a database.
  • a substance having a similar stress . promoter induction/suppression profile as the known or unknown compound is identified.
  • Such identified substance may have an antidote, also referred herein as an antitoxin, i.e. a substance or agent that reduces or represses its toxic activity.
  • antitoxin may also be reducing or repressing the activity of the stress-inducing compound or drug.
  • the stress promoter assay is repeated using only those hosts containing stress promoters, which were induced or suppressed by the stress-inducing compound or drug. Each of those hosts is pre-incubated with varying concentrations of the proposed antitoxin prior to the addition of an inducing/ suppressing concentration of the stress-inducing compound or drug. If pre-incubation with the proposed antitoxin decreases or obliterates the effect of the stress-inducing compound or drug, such an antitoxin will likely be effective.
  • This invention also provides a method of improving active drug design.
  • the present invention relates to a method for identifying and characterizing a stress-inducing compound or drug having a decreased toxicity comprising the steps of: - separately culturing one or more of the above-described host cells,
  • portion of a stress-inducing compound or drug refers to functional group of such compound or drug that is likely to cause cellular damage such as an alteration of normal cellular metabolism, gene expression, translation, or posttranslational modifications in a bacterial cell or population of cells.
  • a new drug is first tested with any of the described kits and methods and its toxicity is determined.
  • the information provided by such methods and kits indicates the cellular mechanism of the drug's toxicity.
  • the particular cellular damage indicated may then be appropriately modified or eliminated depending upon the role that portion or functional group plays in the drug's activity.
  • the resulting modified drug is then retested with the kits and methods of this invention to determine if its toxicity has been sufficiently reduced or eliminated.
  • Drugs improved and modified by this method are also within the scope of this invention
  • a stress-inducing compound or drug can be identified and characterized in the methods according to the invention by direct and/or indirect detection of the assayable product displayed at the surface of the host cell.
  • the assayable gene product can be measured both qualitatively and quantitatively.
  • the stress-induced surface display allows direct qualitative or quantitative assessment, in case of a colored, fluorescent or luminescent protein.
  • the diagnostic kits and methods of this invention also provide the possibility of indirect assessments in particular by using immunolabelling techniques.
  • immunolabelling technique as used herein is meant to refer to various detection methods that use immunoglobulins to detect specific epitopes.
  • immunolabelling technique may comprise but are not limited to ELISA, immunostaining, immunohistochemistry, enzyme immunoassays, Western Blotting, Flow Cytometry, Nephelometry, immunosensors.
  • a frequent problem encountered in existing cellular bio-assays of environmental samples is that signals generated by stress-inducing compounds can be distorted by interfering compounds present in the environmental matrix or the extract thereof.
  • the interfering compounds can interfere directly during signal measurement.
  • turbid samples When turbid samples are tested it becomes difficult to measure the emitted light, color or fluorescence directly from the exposed cells. Due to optical interference from the samples with the optical characteristics of the samples correct readings cannot be obtained.
  • samples are colored due to the presence of natural substances it becomes impossible to measure their effects/toxicity directly with the existing cellular assays.
  • extracts are made from environmental matrices, e.g. using conventional chemical methods such as soxlet extraction, after which these extracts are dried and redissolved in a smaller volume of solvent.
  • cells can be directly added to the matrix and can be distinguished from the matrix after testing due to the highly specific immunological techniques.
  • the soil, water or other matrix can be filtered or centrifuged after which the cells can be fixed on a solid support (e.g. filter, multiwell, etc..) after which they can be detected with the specific immunoglobulin that is specifically targeting the surface exposed protein.
  • a solid support e.g. filter, multiwell, etc..
  • the present invention relates to a method for detecting a stress-inducing compound or drug in a sample comprising the steps of
  • test systems including surface display according to the invention allow (host) cell types that have been transfected to be mixed with other cell types and to perform quantitative assessments on cellular interactions between these host cell types as well as on the impact of the surrounding matrixes on these host cells.
  • hydrophilic compounds such as poly aromatic hydrocarbons, PCBs...
  • PCBs poly aromatic hydrocarbons
  • test system Using a test system according to the present invention, an easier method can be provided for studying and characterizing cellular interactions between these host cell types or the impact of surrounding matrixes on different host cells types.
  • Using a test system according to the present invention it becomes possible to mix various host cell types, each expressing a different reporter gene product into one reaction vessel, without having to use semi ⁇ permeable membranes.
  • Co-culturing or co-exposing cellular surface altered cells is for instance applicable in ecological studies where e.g. interactions among bacterial cell populations and their surrounding matrix can be characterized. This is done for example when one wants to follow the function and location of a specific group of bacteria in biofilms, in sludge and in rhizospheres. By using surface display bacteria it becomes possible to trace back, localize and identify the specific group of cells and evaluate their physiological status.
  • the present invention relates to a method for identifying and characterizing the effects of an environmental matrix or an extract thereof on host cells. Such method comprises the steps of:
  • the present invention relates to a method for identifying and characterizing interactions between different host cell types comprising the steps of;
  • the present invention relates to a method for identifying and characterizing interactions between different host cell types in the presence of one or more stress-inducing compounds or drugs comprising the steps of;
  • kits for performing any of the methods according to the invention.
  • Such kits comprise at least one host cell according to the invention.
  • a stress-inducing compound or drug can be identified and characterized in the diagnostic kits according to the invention by direct and/or indirect detection of the assayable product displayed at the surface of the host cell.
  • the assayable gene product can be measured both qualitatively and quantitatively.
  • the stress-induced surface display allows direct qualitative or quantitative assessment, in case of a colored, fluorescent or luminescent protein.
  • the diagnostic kits and methods of this invention also provide the possibility of indirect assessments in particular by using immunolabelling techniques or flow cytometry.
  • the methods and kits according to the invention are particularly suitable for the identification and evaluation of stress-inducing compounds, which are present in the environment (air, soil, sediments, sludge, water, etc.).
  • stress-inducing compounds which are present in the environment (air, soil, sediments, sludge, water, etc.).
  • Example 1 Non-limiting examples of stress responsive genes of which the toxicologically- inducible promoters are suitable for being fused to and for controlling the expression of a chimeric gene according to the present invention are listed hereunder in Table 1
  • Example 2 Non-limiting examples of E. coli genes comprising transmembrane amino acid sequences which are suitable for use in a construct according to the present invention are listed in Table 2.
  • the chimeric gene comprises the signal sequence, the first 9 amino acids of the mature E. coli Lpp lipoprotein (Genbank accession No V00302) and the sequence encoding the amino acids 46 to 159 of the E. coli membrane protein OmpA.
  • the reporter gene comprises a mutated version of Aequorea victoria green fluorescent protein, gfpmut2 (Cormack et ai, Gene, 173:33-38 1996).
  • a plasmid containing this chimeric gene under control of the Escherichia coli recA promoter (Genbank accession No. V00328; Sancar et ai, 1980) was transformed to E. coli /WC1061 resulting in E. coli strain SD2.
  • the chimeric gene is similar as described above.
  • a plasmid containing this chimeric gene under control of mutated versions of the Escherichia coli recA promoter according to Weisemann and Weinstock (1985) was transformed to E. coli MC1061 resulting in E. coli strains SD3 and SD4.
  • the mutated versions of the promoter (different mutations at different sites of the recA promotor) provide reduced basal expression levels with a factor 10, but preserve the induction ratio and hence allow more reliable observations (analyses) of non-induced versus induced expression of the reporter protein.
  • Table 4 provides a schematic overview of the above-described examples of chimeric genes and their promoters introduced in E. coli.
  • Example 5 Analysis of stress-responsive promoters using surface displayed GFP E. coli strains SD2 were inoculated in LB medium and grown during 16 hours while shaking at 250 rpm until bacteria reached the stationary phase of the growth curve. Bacteria were then diluted (5-fold) and grown further (120 min). The stress inducing compound nalidixic acid was added in a concentration ranging from 0 to 100 ⁇ g/ml and the bacteria were shifted to 25°C. The optical density at 600nm and the fluorescence at 515nm was measured after 2 hours. The fluorescence values demonstrated a clear correlation with the added inducer concentration as indicated in the table 5.
  • the cell density at the highest inducer concentration is decreasing due to lethality effects.
  • This example illustrates that a construct according to the invention can be effectively used to display a reporter gene product at the surface of a host cell in a rapid and easy way in a gradual (progressive) manner over concentrations of a molecule (nalidixic acid) that span several orders of magnitude (at least one) (i.e. from 0 to 100 ⁇ g/ml).
  • the E. coli strains MC1061 used in this example comprise a chimeric gene according to the present invention fused to the toxicologically-inducible micF promoter.
  • the chimeric gene comprises the signal sequence, the first 9 amino acids of the mature E. coli Lpp lipoprotein (Genbank accession No V00302) and the sequence encoding the amino acids 46 to 159 of the E. coli membrane protein OmpA.
  • the reporter gene comprises a mutated version of Aequorea victoria green fluorescent protein, gfpmut2.
  • the E. coli strains MC1061 were inoculated in LB medium and grown during 16 hours while shaking at 250 rpm until bacteria reached the stationary phase of the growth curve. Bacteria were then diluted (5-fold) and grown further (120 min). The stress inducing compound salicylic acid was added in a concentration ranging from 0 to 1000 ⁇ g/ml and the bacteria were shifted to 25°C. The optical density at 600nm and the fluorescence at 515nm was measured after 2 hours. The fluorescence values demonstrated a clear correlation with the added inducer concentration as indicated in the table 6.
  • Example 7 Analysis of stress-responsive promoters using surface displayed GFP
  • the E. coli strains MC1061 used in this example comprise a chimeric gene as described in example 6 fused to the toxicologically-inducible umuDC promoter.
  • the E. coli strains MC1061 were inoculated in LB medium and grown during 16 hours while shaking at 250 rpm until bacteria reached the stationary phase of the growth curve. Bacteria were then diluted (5-fold) and grown further (120 min). The stress inducing compound AZT (3'-azido-3'deoxythymidine) was added in a concentration ranging from 0 to 250 ⁇ g/ml and the bacteria were shifted to 25 0 C. The optical density at 600nm and the fluorescence at 515nm was measured after 2 hours. The fluorescence values demonstrated a clear correlation with the added inducer concentration as indicated in the table 7. Table 7
  • This example illustrates that a construct according to the invention can be effectively used to display a reporter gene product at the surface of a host cell in a rapid and easy way in a gradual (progressive) manner over concentrations of a molecule (AZT) that span several orders of magnitude (at least one) (i.e. from 0 to 250 ⁇ g/ml).
  • Membrane exposed GFP may be measured by fluorescence, as illustrated for instance in examples 5-7.
  • the present example illustrates the detection and measurement of membrane exposed GFP using immunological techniques. Dose dependent determination of membrane exposed GFP was demonstrated using an immunological approach based on a precipitating enzymatic end product substrate.
  • the chimeric gene comprised the signal sequence, the first 9 amino acids of the mature E. coli Lpp lipoprotein and the sequence encoding the amino acids 46 to 159 of the E. coll membrane protein OmpA.
  • the reporter gene comprises a mutated version of Aequorea victoria green fluorescent protein, gfpmut2.
  • a plasmid containing this chimeric gene under control of the E. coli katG promoter was transformed to E. coli MC1061.
  • E. coli cells MC1061 with or without plasmid were grown overnight at 37°C. The culture was diluted 1/3 and grown for another 2 hours at 37 0 C. Menadione was added at different concentrations and the cells were shifted at 25 0 C.
  • the nitrocellulose blot was further incubated for 2 hours at room temperature.
  • the blot was washed with PBS and with Tris buffer (0.1 M Tris, 0.5mM MgCI 2 pH 9.5).
  • the blot was incubated with Tris buffer + NBT/BCIP (final concentration 30 and 15 mg/ml respectively). After 20 minutes the reaction was stopped. Blue dots became visible on the nitrocellulose blot (not shown), and the colour intensities (Table 8) were quantified using standard image analysis software (Sigmascan).
  • the present example illustrates the detection and measurement of membrane exposed GFP using immunological techniques. Dose dependent determination of membrane exposed GFP was demonstrated using an immunological approach based on a soluble enzymatic end product that allows measurement by absorption at 405nm.
  • the chimeric gene comprised the signal sequence, the first 9 amino acids of the mature E. coli Lpp lipoprotein and the sequence encoding the amino acids 46 to 159 of the E. coli membrane protein OmpA.
  • the reporter gene comprises a mutated version of Aequorea victoria green fluorescent protein, gfpmut2.
  • a plasmid containing this chimeric gene under control of the E. coli OsmY promoter was transformed to E. coli MC1061. E. coli strains
  • MC1061 were inoculated in LB medium and grown during 16 hours while shaking at 250 rpm until bacteria reached the stationary phase of the growth curve. Bacteria were then diluted (5- fold) and grown further (120 min). The stress inducing compound NaNO 2 was added in a concentration ranging from 0 to 2500 ⁇ g/ml and the bacteria were shifted to 25°C. After 2 hours these cells were centrifuged and washed in PBS buffer. The washed cells were resuspended in PBS + 2%BSA and used directly in immunological analysis.
  • Both previous examples 8 and 9 illustrate the possibility to use immunologic detection techniques for detecting membrane surface display of a reporter protein in an expression cassette according to the present invention.
  • Example 10 Detection of toxic compounds in soil samples using a system according to the present invention.
  • This example demonstrates the ease of measuring contaminated environmental matrices e.g. soil samples.
  • assays such as the Ames assay are used.
  • assays only allow to test soil samples after chemical extraction of the sample in organic solvents, hereby altering the real availability of contaminants and ultimately incorrectly assessing the risk of pollution (Monarca et al, 2002; Environ. Res 88:64 (2002)).
  • the present invention provides an alternative approach for analysing contaminated soil samples.
  • E.coli strains SD2 were used in this example.
  • the chimeric gene was put under control of the Escherichia coli recA promoter and is inducible by mutagens like e.g. nalidixic acid.
  • About 2 gram of soil sample was taken from a natural uncontaminated area. The samples were dried overnight at 65 0 C. The dried sample was resuspended in water and distributed in different eppendorf tubes, each containing about 100 ⁇ g of soil. The resuspended samples were spiked with nalidixic acid (0; 10; 50; 100ug/ml). Spiking was done on the (in water) resuspended soil samples to have an equal distribution of the inducer on the soil. The spiked samples were dried overnight at 40 0 C to have a sample that is representative for a soil sample.
  • E. coli cells containing the GFP expression cassette behind the recA-promoter (SD2 cells) were grown overnight and diluted 1/3. Cultures were further grown for 2 hours at 37°C, after which the different soil samples were added and the bacteria were further grown at 25°C. After 2hours, the different samples were centrifuged at low speed (500rpm) allowing the soil to be pelleted while the E. coli cells were still present in the soluble phase. GFP Fluorescence of the bacteria was measured. For the different samples a negative control -E. coli with 'empty' plasmid- was always treated in a same way. Results of fluorescence measurements are given in the table 10
  • nalidixic acid The fluorescence measured at 100 ⁇ g/ml of nalidixic acid was lower compared to the fluorescence measured at 75 ⁇ g/ml, probably because of lethal effects of this high nalidixic acid concentration.
  • This example illustrates that a chimeric gene construct according to the invention can be effectively used to display a reporter gene product at the surface of a host cell in a rapid and easy way in a gradual (progressive) manner over concentrations of a molecule (nalidixic acid) that span several orders of magnitude (at least one). Similar dose- response relationships were obtained using western blotting techniques, demonstrating that this assay is robust towards the most commonly interfering confounding factors in cellular environmental screenings (turbidity and coloured samples).
  • a construct according to the invention can be used to display a reporter gene product at the surface of a host cell in a rapid and easy way.
  • a reporter gene product can be targeted and visualized on the host cell surface upon the recognition by the construct of a stress-inducing or mutagen compound. It was demonstrated that a clear dosage effect can be observed using a reporter system according to the invention. It was able to detect a reporter signal within a short time frame.
  • Various detection techniques may be applied for detecting the reporter gene product, including but not limited to physico- chemical measurement techniques such as fluorescence, absorbance, conductivity, magnetic resonance, protein-protein, protein-ligand, receptor-ligand, etc.. immunological techniques or or any other specific measuring technique.
  • the diagnostic kit comprises the following items: 1. Bacterial strains containing a construct according to the present invention including a toxicologically-inducible promoter (e.g. lyophilised or in glycerol stock solutions)
  • a toxicologically-inducible promoter e.g. lyophilised or in glycerol stock solutions
  • Detection medium depending on the reporter system used - comprising: a. Antibodies for immunological detection reactions b. Substrates for the enzymatic reactions if the reporter system is an enzyme
  • Pre-cultures are incubated overnight (e.g. at 37 0 C), after which all bacterial strains reach a stationary phase. 2. Preparation of cells for toxicant exposure
  • a serial dilution of a sample is prepared (e.g. in a 96 multiwell plate). This solution is prepared in the appropriate bacterial growth medium. To every sample the same number of bacterial cells is added.
  • the cellular measurement is performed. This can be either through direct cellular measurement (fluorescence) or indirect through immunological techniques.
  • Toxic responses are reported relative to the control expression level (e.g. as a fold induction). This includes the minimal but existing induction level of unstressed cells.
  • the kit comprises several bacterial strains which all comprise a different construct according to the present invention.
  • several bacterial strains may be used which each comprise one of the following toxicologically-inducible promoters as illustrated in table 11
  • a construct according to the invention can be used to display a reporter gene product at the surface of a host cell in a rapid and easy way.
  • a reporter gene product can be targeted and visualized on the host cell surface upon the recognition by the construct of a stress-inducing or mutagen compound. It was demonstrated that a clear dosage effect can be observed using a reporter system according to the invention. It was possible to detect a reporter signal within a short time frame.
  • Various detection techniques may be applied for detecting the reporter gene product, including but not limited to physico-chemical measurement techniques such as fluorescence, absorbance, conductivity, magnetic resonance, protein-protein, protein-ligand, receptor-ligand, immunological techniques or any other specific measuring technique.

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Abstract

La présente invention concerne un système d’expression biologique présenté en surface destiné à la détection continue de composés générateurs de stress ou toxiques. Le système repose sur l’expression d’un gène chimérique sous le contrôle d’un promoteur inductible d’un point de vue toxicologique dans une cellule hôte procaryote. Dans un autre aspect, la présente invention concerne également un procédé, utilisant ledit système rapporteur biologique, destiné à l’identification et à la caractérisation des composés générateurs de stress. Dans un autre aspect, la présente invention concerne des kits de diagnostic destinés à la réalisation de chacun des procédés selon la présente invention. .
PCT/EP2005/001707 2004-08-18 2005-02-18 Système d’expression presenté en surface WO2006018049A1 (fr)

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WO2018133513A1 (fr) * 2017-01-18 2018-07-26 华南理工大学 Vecteur de détection de substance génotoxique et procédé de détection correspondant

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