EP0943000A1 - Procede tres sensible de detection sur de faibles volumes des atteintes a l'environnement - Google Patents

Procede tres sensible de detection sur de faibles volumes des atteintes a l'environnement

Info

Publication number
EP0943000A1
EP0943000A1 EP97948286A EP97948286A EP0943000A1 EP 0943000 A1 EP0943000 A1 EP 0943000A1 EP 97948286 A EP97948286 A EP 97948286A EP 97948286 A EP97948286 A EP 97948286A EP 0943000 A1 EP0943000 A1 EP 0943000A1
Authority
EP
European Patent Office
Prior art keywords
stress
detector
coli
prokaryote
promoter
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP97948286A
Other languages
German (de)
English (en)
Inventor
Robert Alan Larossa
Pablo Ariel Scolnik
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
EIDP Inc
Original Assignee
EI Du Pont de Nemours and Co
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by EI Du Pont de Nemours and Co filed Critical EI Du Pont de Nemours and Co
Publication of EP0943000A1 publication Critical patent/EP0943000A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters

Definitions

  • the light production reaction catalyzed by luciferase (the product of luxA and luxB), generates light.
  • the energy for light emission is provided by the aldehyde to fatty acid conversion and FMNH 2 oxidation, providing another couple between light production and the cellular energy state.
  • Toxicol, 22(3), 334-8, (1992) describe a test system to assay the levels of HSP70 protein in various species of molluscs and slugs in response to the presence of heavy metals and pesticides. Although the system demonstrated increased levels of HSP70 in response to the presence of Pb 2+ , the technique is cumbersome and lacks sensitivity. A more sophisticated technique described is by Saunders et al. (WO 9002947). The Saunders et al. technique involves detecting increased levels of HSP60 and HSP70 in organisms exposed to pollutants in an aqueous environment.
  • transformation refers to the acquisition of new genes in a cell after the incorporation of nucleic acid.
  • stress response refers to the cellular response resulting in the induction of either detectable levels of stress proteins or in a state more tolerant to exposure to another insult or an increased dose of the environmental insult.
  • the invention also provides a transformation vector containing a stress inducible promoter-/ux gene fusion, capable of transforming a bacterial host cell for the expression of the Lux proteins.
  • a transformation vector containing a stress inducible promoter-/ux gene fusion capable of transforming a bacterial host cell for the expression of the Lux proteins.
  • a variety of transformation vectors may be used, however, those capable of transforming E. coli are preferred.
  • pGrpELux.3, pGrpELux.5, pRYOOl, pRY002. and pRY006 are five specific examples of suitable transformation vectors whose construction is given in detail in the following text. These vectors represent only a sample of the total number of vectors created for the purpose of introducing stress promoter-/wx reporter fusions into host cells.
  • Figures 6a and 6b illustrate the sensitivity of the transformants WM1021 and WM1026 (containing the dnaK promoter lux fushion) to the stress of varying concentrations of ethanol. It is interesting to note that at the sublethal concentrations of ethanol varying from 1%> to 4%, light emission increased in a fashion similar to the TV1060 cultures. By contrast, lethal concentrations of ethanol in the ranges of 8% to 16%> produced a decrease in light emission from the detector cultures. Likewise, higher concentrations of PCP also result in decrease of light output in strain TV 1076 ( Figure 7). Thus, it is evident that the method of the present invention is capable of a bi-modular function.
  • EXAMPLE 4 Stress induction of bioluminescence at 4%o ethanol Strain TV 1060 was grown at 37 °C in LB medium containing kanamycin (25 ⁇ g/mL) until it reached Klett 56 (measured on a Klett-Summerson colorimeter with a #66 red filter) at which time it was diluted 1 :11 into the same medium at ambient temperature and allowed to grow for 3 h at ambient temperature until reaching a density of 20 Klett Units. 100 ⁇ L of cells were placed into the wells of a microtiter plate followed by the addition of either 10 ⁇ L 40%) ethanol (experimental, final concentration 4% ethanol) or 10 ⁇ L of distilled water (control).
  • EXAMPLE 5 Stress induction of bioluminescence at varying concentrations of ethanol Strain TV 1060 was grown overnight in LB medium containing kanamycin (25 ⁇ g/mL) and then diluted 1 : 100 in the same medium and grown at room temperature until reaching a Klett of 20. 100 ⁇ L of cells were placed into the wells of a microtiter plate containing 100 ⁇ L of either 2%>, 4%>, 8%> or 16%. (giving final concentrations of 1%, 2%, 4%, and 8%) respectively of ethanol, experimental) or 100 ⁇ L of the same medium (control). The plate was immediately placed into a luminometer, model ML3000 (Dynatech Laboratories.
  • EXAMPLE 22 Construction and Response of fabA transformed host cell to fattv acid starvation
  • the fabA gene encodes the enzyme responsible for the placement of a double bond in the fatty acids and hence membrane of £ coli. Such double bonds are an absolute requirement for growth.
  • Synthesis of fabA is directed by two promoter elements: a low level, constitutive upstream and an inducible downstream promoter. The location of the two promoters in the sequence surrounding fabA has been determined.
  • the PCR primers shown in Table IV are designed to allow cloning of the inducible downstream promoter without the constitutive upstream promoter.
  • E. coli host RFM443 is transformed with the pFabALux is grown overnight in minimal E medium containing kanamycin (10 ⁇ g/mL) at 29 °C.
  • the overnight cultures are diluted and grown to early log phase in the same media as the overnight culture.
  • Culture turbidity is measured with a Klett-Summerson colorimeter with a #66 red filter.
  • Luminescence present in 50 ⁇ L of cell culture in the presence or absence of 50 ⁇ g/mL of sulfometuron methyl is quantitated in a Dynatech ML3000 luminometer at 26 °C. It is seen that cultures in the presence of sulfometuron methyl demonstrate a 10-25 fold increase in luminesces when compared with cultures in the absence of sulfometuron methyl.
  • MOLECULE TYPE DNA (genomic)

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Il est possible de détecter et mesurer d'infimes variations des contraintes environnementales à des niveaux sublétaux sous forme d'une réponse générique auxdites contraintes. L'invention porte sur un procédé amélioré de détection des susdites variation dans de très faibles volumes, ce qui permet des rendements d'analyse très élevés. Les variations des contraintes sont mises en évidence par des modifications de la luminescence d'un micro-organisme produit par génie génétique. Le complexe de gènes luminescents est commandé par un promoteur réagissant aux contraintes. Les volumes prévus vont d'environ 0,5 uL à environ 10 mL du milieu dans lequel le micro-organisme détecteur est en suspension.
EP97948286A 1996-11-15 1997-11-14 Procede tres sensible de detection sur de faibles volumes des atteintes a l'environnement Withdrawn EP0943000A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US3127296P 1996-11-15 1996-11-15
US31272P 1996-11-15
PCT/US1997/020793 WO1998021347A1 (fr) 1996-11-15 1997-11-14 Procede tres sensible de detection sur de faibles volumes des atteintes a l'environnement

Publications (1)

Publication Number Publication Date
EP0943000A1 true EP0943000A1 (fr) 1999-09-22

Family

ID=21858542

Family Applications (1)

Application Number Title Priority Date Filing Date
EP97948286A Withdrawn EP0943000A1 (fr) 1996-11-15 1997-11-14 Procede tres sensible de detection sur de faibles volumes des atteintes a l'environnement

Country Status (4)

Country Link
EP (1) EP0943000A1 (fr)
JP (1) JP2001503990A (fr)
CA (1) CA2267999A1 (fr)
WO (1) WO1998021347A1 (fr)

Families Citing this family (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IL131336A0 (en) * 1997-02-28 2001-01-28 Du Pont A method for identifying the site of action of xenobiotic chemicals
KR100414784B1 (ko) * 2000-01-18 2004-01-13 (주)바이오니아 연속식 수질 독성 검사 장치
GB2364307A (en) * 2000-04-03 2002-01-23 Univ Surrey Bioluminescent reporters
US20030108980A1 (en) * 2000-08-14 2003-06-12 Sayler Gary S. Bioluminescent methods for direct visual detection of environmental compounds
CA2365791A1 (fr) 2000-12-22 2002-06-22 Pfizer Products Inc. Essais par bioluminescence nouveaux et souches bacteriennes utiles a cet egard
AU2003277276A1 (en) 2002-10-04 2004-05-04 Genencor International, Inc. Improved production of bacterial strains cross reference to related applications
CN100334431C (zh) * 2003-02-20 2007-08-29 华为技术有限公司 一种环境应力实验自动测试方法
WO2004076685A2 (fr) * 2003-02-28 2004-09-10 Lux Biotechnology Limited Biocapteur et bioanalyse fongique
JP2008035819A (ja) * 2006-08-09 2008-02-21 Daikin Ind Ltd プロモーターアッセイ方法に用いる容器、該容器を含むプレートおよびそれらを用いるプロモーターアッセイ方法
US20140051101A1 (en) * 2012-08-20 2014-02-20 Carnegie Institution Of Washington Luciferase Reporter System for Roots and Methods of Using the Same

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100300932B1 (ko) * 1992-07-06 2001-10-22 조이스 브린톤 리포터유전자에융합된세균의스트레스프로모터를이용하여독성을측정하는방법및진단키트
IL107815A (en) * 1992-12-04 2003-12-10 Du Pont Genetic constructs comprising a stress-responsive promoter linked to a lux reporter operon and methods of use in environmental testing
DE69413491T2 (de) * 1993-10-22 1999-03-11 Toyoda Chuo Kenkyusho Kk Verfahren zum Nachweis von Mutagenen unter Verwendung eines Lumineszensgens
ATE222605T1 (de) * 1994-11-23 2002-09-15 Du Pont Bioluminiscente lyophilisierte bakterien-reagenz zur giftstoff nachweit

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9821347A1 *

Also Published As

Publication number Publication date
JP2001503990A (ja) 2001-03-27
CA2267999A1 (fr) 1998-05-22
WO1998021347A1 (fr) 1998-05-22

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