WO1990004053A1 - Dispositif d'electropolissage de surfaces - Google Patents
Dispositif d'electropolissage de surfaces Download PDFInfo
- Publication number
- WO1990004053A1 WO1990004053A1 PCT/DE1988/000626 DE8800626W WO9004053A1 WO 1990004053 A1 WO1990004053 A1 WO 1990004053A1 DE 8800626 W DE8800626 W DE 8800626W WO 9004053 A1 WO9004053 A1 WO 9004053A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- bioluminescence
- gene
- microorganisms
- housing
- indicator
- Prior art date
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C25—ELECTROLYTIC OR ELECTROPHORETIC PROCESSES; APPARATUS THEREFOR
- C25F—PROCESSES FOR THE ELECTROLYTIC REMOVAL OF MATERIALS FROM OBJECTS; APPARATUS THEREFOR
- C25F7/00—Constructional parts, or assemblies thereof, of cells for electrolytic removal of material from objects; Servicing or operating
-
- G—PHYSICS
- G21—NUCLEAR PHYSICS; NUCLEAR ENGINEERING
- G21F—PROTECTION AGAINST X-RADIATION, GAMMA RADIATION, CORPUSCULAR RADIATION OR PARTICLE BOMBARDMENT; TREATING RADIOACTIVELY CONTAMINATED MATERIAL; DECONTAMINATION ARRANGEMENTS THEREFOR
- G21F9/00—Treating radioactively contaminated material; Decontamination arrangements therefor
- G21F9/001—Decontamination of contaminated objects, apparatus, clothes, food; Preventing contamination thereof
- G21F9/002—Decontamination of the surface of objects with chemical or electrochemical processes
- G21F9/004—Decontamination of the surface of objects with chemical or electrochemical processes of metallic surfaces
Definitions
- the invention relates to a method for the detection of toxic substances in a liquid or gaseous environment with the help of specifically sensitive and / or resistant microorganism strains which
- All microorganisms are capable of emitting light (bio-luminescence) by introducing a plasmid-encoded luciferase gene.
- bioluminescence of the indicator organisms enables changes in their metabolism or the loss of their viability as an effect of a toxic substance in the test medium to be detected quickly and with great sensitivity.
- microorganisms for measuring critical loads caused by toxic substances (US Pat. No. 3,981,777) and for determining antibiotic concentrations (EP. No. 0200 226) has been established.
- the applications are based on the detection of the growth of the test organisms by counting colonies, turbidity measurements, nephelometry, etc.
- These measuring methods require the cultivation of large amounts of microorganisms, so that reliable measurements are only possible after can be carried out over a period of 16 to 72 hours. In contrast, the method described below allows the evaluation of measurement results after less than 2 hours.
- Naturally occurring bioluminescent microorganisms are of marine origin and therefore require a high ionic strength in the test medium.
- the production of the required osmolarity means a possibly falsifying intervention in the measurement.
- the invention described here makes the methods ex- use experimental genetics to transform in principle any desired bacterial strain by introducing a specially constructed plasmid vector (pGL3, see below) into an organism capable of bioluminescence. While the principle of transforming a bacterial strain with a plasmid-encoded luciferase LUX gene has already been described (US Pat. No. 4,581,335), the present invention represents a decisive improvement: the expression of the LUX gene and the ability to bioluminescence is no longer permanent (constitutive), but can be switched on and off in a temperature-dependent manner.
- the feature of adjustability is achieved by construction of a plasmid Vek ⁇ catalyst which both the luciferase gene complex of Vi • brio harveyi (lux genes A and B), and the C allele Ig57 of phage lambda repressor gene ( contains thermolabile gene product1).
- the inducible bioluminescence achieved by this concept has two important advantages compared to permanently light-emitting test organisms:
- the signal to noise ratio of the individual measurement is improved.
- Another characteristic of the development according to the invention is the possibility of introducing additional resistance or hypersensitivity to specific toxic mediating genes in the plasmid vector outlined; this is made possible by molecular cloning of a corresponding gene into existing specific restriction endonuclease sites.
- Measurement results take about ten times less time than conventional microbiological cultivation and measurement methods and
- any suitable, recommended or prescribed in standardized reference tests bacterial strain for bioluminescence measurements can be set up.
- E.coli ATCC 25922 may be mentioned as an example of a bacterial strain which belongs to the latter category; this strain is the official (WHO) reference strain for standardized antibiotic inhibition tests.
- E. coli ATCC 25922 and other strains frequently used in clinical and industrial tests (E. coli K- and B; chi 1776 etc.) have been successfully converted by the applicant into the species capable of bioluminescence by introducing the plasmid pGL3 .
- the regulation takes place by using the allele 857 of the lambda repressor in a temperature-dependent manner.
- the repressor protein By increasing the temperature to above 37 ° C, the repressor protein is inactivated and the LUX gene complex is released for transcription in mRNA, which ultimately results in an increase in the bioluminescence activity of the indicator organism by more than three orders of magnitude.
- the difference in the size of the bioluminescence signal between the repressed and transcribed state of the LUX gene complex - and thus the signal-to-noise ratio of the measurements - is more than a factor of ten in test organisms equipped with pGL3 better than those which are induced to bioluminescence by chemical induction of the lacZ gene (cf. US Pat. No. 4,581,335).
- the plasmid pGL3 which was designed for the generation of controllable bioluminescence in microorganisms, was constructed by using conventional molecular biological cloning techniques; it contains:
- Luciferase gene complex (LUX A; LUX B) from Vibrio Harvey
- the LUX-gene complex is 3 ', ie "behind" promoter. J the P Loo defined and is therefore transcribed therefrom; the P R_M ..- pro- For its part, motor is negatively controlled by the gene product of the C gene - the lambda repressor; Although this lambda repressor is always synthesized in sufficient quantity by constitutive expression of the P_-dependent C ⁇ gene, it is heat-labile by using the C Ig57 allele.
- the P_ M promoter remains blocked by binding the repressor until the repressor protein is denatured by increasing the temperature to 37 ° to 42 ° C for 5 to 15 minutes and the P _-.- promotor falls off.
- pGL3 The DNA-dependent RNA polymerase then binds to the free promoter and causes the massive transcription of the LUX gene complex. Overall, this construction of pGL3 leads to an adjustable, since temperature-dependent synthesis of the enzyme luciferase, which is responsible for the bioluminescence of the indicator organisms.
- the construction base of pGL3 is the plasmid pLK915 (see KK Stanley, and JP Luzio, EMBO J. 3: 1429 to 1434, 1984).
- Compatibility of the Sal 1 interface with Bam Hl was determined by previously attaching the 45 base pair long Bam Hl - Sal 1 restriction fragment from the polylinker region of the plasmid "pBluescript" (company "STRATAGENE”; San Diego, Cal., USA ) at the Sal 1 end and subsequent restriction cleavage with the enzyme Bam HI.
- the above-described plasmid pGL3 can in principle be introduced into any desired species of microorganism by standard techniques in order finally to be cultured in large quantities. It is possible to store bacteria as glycerol cultures at -20 ° C or after freeze-drying at ambient temperature. Freeze-drying is carried out either in glass ampoules or in specially made plastic containers, e.g. in two separate chambers the lyophilized microorganisms or the components necessary for the revitalization of the bacteria. Contain nutrient medium. In order to carry out measurements of toxic substances in a gas phase, the indicator organisms are permanently separated from a measuring compartment by a membrane impermeable to water vapor. In this way, the constant composition of the growth medium and thus the measurement over a longer measurement period, as required for toxicity tests of gases, is ensured.
- the application of the lyophilized indicator organisms to a suitable carrier material in the form of a test strip is a preferred form of application.
- the microorganisms are applied to the carrier matrix in a defined amount, fixed by a special process, freeze-dried and finally sealed as a test strip.
- the indicator bacteria are incubated for one to two hours in aqueous nutrient medium Ambient temperature revitalized.
- the measuring principle of the toxicity test described here is based on the use of a two-step method:
- a "screening" is carried out with a number of different test organisms of defined sensitivity for the detection of general toxicity in a sample. Thereafter, further detection reactions can be carried out with strains which are provided with a specific, genetically present or introduced by transformation property of resistance or (hyper) sensitivity.
- a toxin to which a strain is specifically resistant will lead to an un or only partially reduced bioluminescence signal; in contrast, a non-resistant control strain is more serious in its viability and, correlated with it, its ability to bioluminescence.
- the measuring method according to the invention is therefore suitable on the one hand for determining the degree of generally present toxicity of a sample through the growth behavior of microorganisms, and on the other hand it is also possible to use all substances with which a specific reactivity of certain test agents ganisms exists to identify and determine their concentration approximately easily and quickly.
- indicator organisms - this time endowed with specific resistance or sensitivity to certain toxic substances - are incubated with the sample liquid in order to finally carry out bioluminescence measurements. If the bioluminescence signal is unchanged only in one of the test organisms, for example at the level of the control value, " while all other microorganisms experience a reduction in measurable bioluminescence, the toxin to which the strain in question is resistant is identified.
- a strain of the gram-negative bacterium E. coli ATCC 25922 was treated with a plasmid [(pGL3.T, which in addition to the grain components of pGL3 (see above) contains a tetracycline resistance gene (strain "T” in Fig. 2)].
- pGL3.T which in addition to the grain components of pGL3 (see above) contains a tetracycline resistance gene (strain "T” in Fig. 2)].
- strain "C" a derivative of ATCC 25922 - equipped with pGL3 - was used.
- Several 0.5 ml samples of raw milk with different concentrations of the antibiotic tetracycline were mixed with 0.1 ml (about 2 x 10 bacteria) suspensions of the strains C and T and incubated for 20 minutes at room temperature. After heating the cultures at 40 ° C.
- FIG. 1 shows the measurement results of samples with five different antibiotic concentrations as relative light signals (RLU; ordinate) as a function of time.
- the bioluminescence signal of the tetracycline-resistant strain T is relatively constant over the measurement period, while strain C shows a decrease in the amount of light emitted in a concentration-dependent manner.
- strain C shows a decrease in the amount of light emitted in a concentration-dependent manner.
- the fact that the growth properties of the indicator strain T are not significantly impaired by tetracycline makes it possible to use this antibiotic in samples (e.g. milk) of unknown origin.
- the determination of absolute concentrations of this antibiotic or other toxic substances against which an indicator strain is specifically resistant is possible to a good approximation by including reference values of known concentration of the toxin in the measurement series.
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- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Electrochemistry (AREA)
- Organic Chemistry (AREA)
- Metallurgy (AREA)
- Materials Engineering (AREA)
- General Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- General Engineering & Computer Science (AREA)
- High Energy & Nuclear Physics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Cleaning In General (AREA)
Abstract
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE8888908666T DE3878697D1 (de) | 1988-10-10 | 1988-10-10 | Vorrichtung zum elektropolieren von oberflaechen. |
PCT/DE1988/000626 WO1990004053A1 (fr) | 1988-10-10 | 1988-10-10 | Dispositif d'electropolissage de surfaces |
EP88908666A EP0436528B1 (fr) | 1988-10-10 | 1988-10-10 | Dispositif d'electropolissage de surfaces |
US07/683,261 US5135632A (en) | 1988-10-10 | 1991-04-10 | Apparatus for electropolishing surfaces |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PCT/DE1988/000626 WO1990004053A1 (fr) | 1988-10-10 | 1988-10-10 | Dispositif d'electropolissage de surfaces |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1990004053A1 true WO1990004053A1 (fr) | 1990-04-19 |
Family
ID=6819755
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/DE1988/000626 WO1990004053A1 (fr) | 1988-10-10 | 1988-10-10 | Dispositif d'electropolissage de surfaces |
Country Status (3)
Country | Link |
---|---|
EP (1) | EP0436528B1 (fr) |
DE (1) | DE3878697D1 (fr) |
WO (1) | WO1990004053A1 (fr) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998022953A1 (fr) * | 1996-11-15 | 1998-05-28 | Babcock Rosyth Defence Limited | Limitation de la contamination de la surface d'un element metallique par des radionucleides |
US6340572B1 (en) | 1991-04-04 | 2002-01-22 | Board Of Regents, The University Of Texas System | Kit for the isolation, identification and quantitation of toxicants |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
IT1311147B1 (it) * | 1999-11-04 | 2002-03-04 | Edk Res Ag | Macchina per pulizia localizzata con cella, elettrolitica e/o adultrasuoni, di decapaggio e/o lucidatura |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0146833A2 (fr) * | 1983-12-14 | 1985-07-03 | Siemens Aktiengesellschaft | Dispositif pour le polissage électrolytique des surfaces intérieures d'objets cylindriques creux |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3370175A (en) * | 1965-01-28 | 1968-02-20 | North American Rockwell | Toxicant detector |
CA1103050A (fr) * | 1977-09-28 | 1981-06-16 | Anthony A. Bulich | Methode de detection de substances toxiques dans un liquide |
US4581335A (en) * | 1982-12-01 | 1986-04-08 | Texas A&M University System | Process for producing a cloned luciferase-synthesizing microorganism |
DE3789766T2 (de) * | 1986-07-22 | 1994-09-29 | Texas A & M University Syst | Verwendung von bakteriellen luciferase strukturellen genen zum klonieren und zur steuerung der genexpression in mikroorganismen, sowie zur etikettierung und identifizierung von genetisch gebildeten organismen. |
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1988
- 1988-10-10 EP EP88908666A patent/EP0436528B1/fr not_active Expired - Lifetime
- 1988-10-10 DE DE8888908666T patent/DE3878697D1/de not_active Expired - Fee Related
- 1988-10-10 WO PCT/DE1988/000626 patent/WO1990004053A1/fr active IP Right Grant
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0146833A2 (fr) * | 1983-12-14 | 1985-07-03 | Siemens Aktiengesellschaft | Dispositif pour le polissage électrolytique des surfaces intérieures d'objets cylindriques creux |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6340572B1 (en) | 1991-04-04 | 2002-01-22 | Board Of Regents, The University Of Texas System | Kit for the isolation, identification and quantitation of toxicants |
WO1998022953A1 (fr) * | 1996-11-15 | 1998-05-28 | Babcock Rosyth Defence Limited | Limitation de la contamination de la surface d'un element metallique par des radionucleides |
Also Published As
Publication number | Publication date |
---|---|
EP0436528B1 (fr) | 1993-02-24 |
EP0436528A1 (fr) | 1991-07-17 |
DE3878697D1 (de) | 1993-04-01 |
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