DE4103289A1 - Determn. of lactose repressor over-producing microorganisms - using a phage with a marker gene contg. C-terminal with a lactose operator and promoter which forms an anti-sense RNA of the marker - Google Patents
Determn. of lactose repressor over-producing microorganisms - using a phage with a marker gene contg. C-terminal with a lactose operator and promoter which forms an anti-sense RNA of the markerInfo
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- DE4103289A1 DE4103289A1 DE4103289A DE4103289A DE4103289A1 DE 4103289 A1 DE4103289 A1 DE 4103289A1 DE 4103289 A DE4103289 A DE 4103289A DE 4103289 A DE4103289 A DE 4103289A DE 4103289 A1 DE4103289 A1 DE 4103289A1
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- lactose
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- 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
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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- 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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
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- 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
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y113/00—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13)
- C12Y113/11—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13) with incorporation of two atoms of oxygen (1.13.11)
- C12Y113/11001—Catechol 1,2-dioxygenase (1.13.11.1)
Abstract
Description
Jeder Umgang mit Mikroorganismen, auch mit gentechnisch verän derten, ist mit dem Risiko der unbeabsichtigten Freisetzung dieser Mikroorganismen verbunden. In der Diskussion um die Risi ken und Gefahren des Umgangs mit gentechnisch veränderten Orga nismen nimmt die Frage des Nachweises gentechnisch veränderter Mikroorganismen in der Umwelt daher eine zentrale Stellung ein. Der Nachweis spezifischer Mikroorganismen kann z. B. dazu dienen, ein mögliches Entweichen von Mikroorganismen bei Produktionspro zessen zu überwachen. In solchen Fällen ist es wichtig, über spezifische Nachweismethoden zu verfügen, da generelle Verfahren wegen der ubiquitären Anwesenheit von Mikroorganismen nicht aus sagefähig genug sind.Any handling of microorganisms, including genetically modified ones is associated with the risk of accidental release linked to these microorganisms. In the discussion about the risks and dangers of dealing with genetically modified org nisms takes the question of the detection of genetically modified Microorganisms therefore play a central role in the environment. The detection of specific microorganisms can e.g. B. serve a possible escape of microorganisms in production pro monitoring. In such cases it is important to think about to have specific detection methods as general procedures due to the ubiquitous presence of microorganisms are able to say enough.
Bei der Produktion mit gentechnisch veränderten Mikroorganismen, deren Expressionssystem auf dem Laktose-Operon von E.coli beru hen, werden spezifische Stämme verwendet. Allen Stämmen ist ge meinsam, daß sie zur Verbesserung der Stabilität des Expres sionsstammes eine die Überproduktion des Laktose-Repressor-Pro teins bewirkende Mutation tragen, wodurch das Regulationssystem stabilisiert und von außen regulierbar wird. Diese Mutation wird künstlich in E.coli eingeführt, sie kommt in Wild-Typ-Stämmen nicht vor (B. Müller-Hill et al., Proc. Natl. Acad. Sci. USA 59, 1259 (1968)).When producing with genetically modified microorganisms, whose expression system is based on the lactose operon from E. coli hen, specific strains are used. All tribes are together that they improve the stability of the express strain of overproduction of the lactose repressor pro teins causing mutation, causing the regulatory system is stabilized and regulated from the outside. This mutation will Artificially introduced in E.coli, it occurs in wild-type strains not before (B. Müller-Hill et al., Proc. Natl. Acad. Sci. USA 59, 1259 (1968)).
Die Erfindung ist demnach auf ein Verfahren zum Nachweis von den Laktose-Repressor überproduzierenden Mikroorganismen gerichtet, das dadurch gekennzeichnet ist, daß man Mikroorganismen, bei de nen die Anwesenheit von den Laktose-Repressor überproduzierenden Stämmen vermutet wird, mit Markergenen aufweisenden Bakteriopha gen infiziert, wobei den Bakteriophagen-Markergenen C-terminal ein Laktose-Promotor/Operator angehängt ist, der in Gegenrich tung (anti-sense) in das Markergen unter Bildung einer anti sense RNA des Markergens transkribiert, und das man die Anwesen heit von Expressionsprodukten des Markergens nachweist. Handelt es sich bei den zu untersuchenden Stämmen um solche des Wild-Typs bzw. andere Stämme, die keine den Laktose-Repressor überproduzierende Mutation aufweisen, erfolgt die Bildung der anti-sense-RNA und es sind demgemäß keine Expressionsprodukte des Markergens nachweisbar.The invention is therefore based on a method for the detection of Directed to lactose repressor overproducing microorganisms, which is characterized in that microorganisms in de NEN the presence of the lactose repressor overproducing Strains is suspected with bacteriopha containing marker genes gene infected, the bacteriophage marker gene C-terminal a lactose promoter / operator is attached, which is in opposite direction anti-sense in the marker gene to form an anti sense RNA of the marker gene is transcribed, and that one the property evidence of expression products of the marker gene. If the strains to be examined are those of the Wild-type or other strains that do not use the lactose repressor overproducing mutation, the formation of anti-sense RNA and, accordingly, are not expression products of the marker gene is detectable.
Gemäß einer bevorzugten Ausführungsform der Erfindung wird als Markergen das xylE-Gen von Pseudomonas putida oder ein Leuchtgen von Vibrio fischeri verwendet.According to a preferred embodiment of the invention, as Marker gene, the xylE gene from Pseudomonas putida or a luminous gene used by Vibrio fischeri.
Gemäß einer weiteren bevorzugten Ausführungsform der Erfindung werden als Bakteriophage der Phage Lambda oder Derivate dessel ben verwendet.According to a further preferred embodiment of the invention are called bacteriophages of phage lambda or derivatives thereof ben used.
Die Erfindung beruht auf der Erkenntnis, daß die Information von Genen, von denen neben der mRNA eine anti-sense RNA gebildet wird, nicht in ein Protein übersetzt werden kann, da die Inter aktion der mRNA und der anti-sense RNA, die zueinander komple mentär sind, eine Translation an den Ribosomen verhindert. Die gleichzeitige Bildung von mRNA und anti-sense RNA eines Marker gens wird dadurch erreicht, daß am C-terminalen Ende des am N terminalen Ende einen Promotor aufweisenden Markergens ein Laktose-Promotor/Operator angehängt wird, der in Gegenrichtung (anti-sense) transkribiert. Erfindungsgemäß wird aus einem geeigneten Plasmid mit Hilfe von Restriktionsenzymen ein geeig netes Markergen isoliert und in ein zweites Plasmid kloniert, das den Laktose-Promotor/Operator und eine Lac-alpha-Region auf weist und das mit denselben Restriktionsenzymen restringiert wird, bei dem aber die Reihenfolge der Restriktionsstellen ver tauscht ist. Durch die unterschiedliche Position der Restrik tionsschnittstellen in den beiden Plasmiden gerät die Transkrip tionsrichtung des Markergens in Opposition zu der des Laktose- Promotors des zweiten Plasmids. Von dem klonierten Plasmid wird das Markergen mit geeigneten Methoden auf einen Phagen übertra gen, der die Produktionsstämme infizieren kann, z. B. auf den Phagen Lambda. The invention is based on the knowledge that the information from Genes, of which an anti-sense RNA is formed in addition to the mRNA cannot be translated into a protein because the Inter action of the mRNA and the anti-sense RNA, which complete each other are mental, translation on the ribosomes is prevented. The simultaneous formation of mRNA and anti-sense RNA of a marker gene is achieved in that at the C-terminal end of the N terminal end of a promoter-containing marker gene Lactose promoter / operator is attached in the opposite direction (anti-sense) transcribed. According to the invention suitable plasmid using restriction enzymes isolated marker gene isolated and cloned into a second plasmid, which has the lactose promoter / operator and a Lac alpha region points and that restricts with the same restriction enzymes is, but in which the order of the restriction sites ver is exchanged. Due to the different position of the restriction The transcription ends up in the two plasmids direction of the marker gene in opposition to that of the lactose Promoter of the second plasmid. From the cloned plasmid transfer the marker gene to a phage using suitable methods gene that can infect the production strains, e.g. B. on the Phage lambda.
In normalen E.coli-Stämmen, die keine den Laktose-Repressor überproduzierende Mutation tragen, findet keine Expression des Markergens statt. In einem den Laktose-Repressor überproduzie renden Stamm, z. B. einem von dem Laktose-Operator abgeleiteten Produktionsstamm, wird durch Repression des anti-sense-Promo tor/Operators der Anteil der anti-sense RNA zugunsten der mRNA verringert, so daß das entsprechende Genprodukt synthetisiert wird und nachgewiesen werden kann.In normal E. coli strains that do not use the lactose repressor carry overproducing mutation, finds no expression of the Marker gene instead. Overproduce the lactose repressor renden tribe, e.g. B. one derived from the lactose operator Production strain, is by repression of the anti-sense promo tor / operator the proportion of anti-sense RNA in favor of mRNA reduced so that the corresponding gene product is synthesized will and can be proven.
Als gut zu identifizierendes Markergen werden Gene verwendet, die in der Natur nicht weit verbreitet sind, z. B. das xylE-Gen von Pseudomonas putida oder die Leuchtgene von Vibrio fischeri.Genes are used as the easily identifiable marker gene, which are not widespread in nature, e.g. B. the xylE gene from Pseudomonas putida or the luminous genes from Vibrio fischeri.
Die Erfindung wird im folgenden anhand eines bevorzugten Ausfüh rungsbeispiels näher erläutert.The invention is based on a preferred embodiment example explained in more detail.
Ausgegangen wird von dem in der EP 01 46 572 beschriebenen Plasmid pMM8, das den Laktose-Promotor/Operator, eine Lac-alpha- Region und die Multicloning-Sequenz des Plasmids pUR250 (U. Rüther et al. Nucl. Acids Res. 10 (1982), 5765) aufweist, wobei anstelle des Plasmids pMM8 ebenso das Plasmid pUR250 verwendet werden kann.The starting point is that described in EP 01 46 572 Plasmid pMM8, which is the lactose promoter / operator, a Lac-alpha Region and the multicloning sequence of the plasmid pUR250 (U. Rüther et al. Nucl. Acids Res. 10 (1982), 5765), the plasmid pUR250 being used instead of the plasmid pMM8 can be used.
Das xylE-Gen von Pseudomonas putida wurde mit gebräuchlichen Methoden (T. Maniatis, E.F. Fritsch und J. Sambrook, Molecular Cloning, Gold Spring Harbor; New York 1982) als BamHI/HindIII- Fragment aus dem Plasmid pEPA53 (S.M. Cuskey & A.B. Sprenkle, J. Bacteriol. 170 (1988) 3742) isoliert und in die Lac-alpha-Region des Plasmids pMM8 insertiert, das mit den gleichen Enzymen re stringiert war. Das resultierende Plasmid wurde pFM24 genannt. Aufgrund der unterschiedlichen Position der BamHI- und HindIII- Restriktionsschnittstellen in den Plasmiden pMM8 und pEPA53 gerät die Transkriptionsrichtung des xylE-Gens in Opposition zu der des Laktose-Promotors in dem Plasmid pMM8. The xylE gene from Pseudomonas putida has been used with common Methods (T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning, Gold Spring Harbor; New York 1982) as BamHI / HindIII- Fragment from the plasmid pEPA53 (S.M. Cuskey & A.B. Sprenkle, J. Bacteriol. 170 (1988) 3742) isolated and in the Lac alpha region of the plasmid pMM8 inserted with the same enzymes right was stringed. The resulting plasmid was named pFM24. Due to the different position of the BamHI and HindIII Restriction sites in the plasmids pMM8 and pEPA53 the direction of transcription of the xylE gene is in opposition that of the lactose promoter in the plasmid pMM8.
Das Plasmid pFM24, in einen Wild-Typ Stamm transformiert, bildet nach Besprühen mit Gatechol weiße Kolonien, ebenso ein den Laktose-Repressor überproduzierender Stamm nach Wachstum auf Agrarplatten mit Induktor, wodurch der Laktose-Repressor inakti viert wird und Verhältnisse wie in einem Wild-Typ Stamm vorlie gen. In einem den Laktose-Repressor überproduzierenden Stamm, auf Agrarplatten ohne Induktor gewachsen, färben sich die Kolo nien nach Besprühen mit Gatechol gelb.The plasmid pFM24, transformed into a wild-type strain, forms after spraying with Gatechol white colonies, as well as the Lactose repressor overproducing strain after growth Agricultural plates with an inductor, making the lactose repressor inactivated fourth and conditions as in a wild-type strain in a strain overproducing the lactose repressor, grown on agricultural plates without an inductor, the Kolo color after spraying with Gatechol yellow.
Das Plasmid pFM24 wurde in den E.coli-Stamm 50-1 (M. Mieschendahl, Dotorarbeit, Universität Köln, 1981) transfor miert. Stamm 50-1 ist der E.coli-Stamm GSH50 (J. Miller, Experi nents in Molecular Genetics, Gold Spring Harbor, New York 1972), der lysogen für den Lambda-Phagen Lambda placs ist (K. Ippen et al., J. Bacteriol. 108, 5, 1971). Dieser Phage weist Teile des Lac-Operons einschließlich des Laktose-Promotors und des lacZ- Gens auf. Die lac-Sequenzen im Plasmid pMM8 sind homolog zu die sen lac-Sequenzen des Phagen Lambda plac5. Durch Bildung von Homogenoten (J. Miller, s. o.) kam es zur Rekombination der lac- Region des Plasmids pFM24 und der lac-Region des Phagen Lambda plac5, wodurch das xylE-Gen vom Plasmid auf den Phagen übertra gen wurde. Dadurch wurde gleichzeitig das lacZ-Gen des Phagen inaktiviert, so daß die gesuchten Homogenoten als weiße Kolonien auf x-gal-Indikatorplatten isoliert werden konnten. X-gal ist ein farbloses 5-Brom-4-chlor-3-indolyl-beta-galaktosid, aus dem nach Spalten durch beta-Galaktosidase, dem Genprodukt des lacz- Gens, ein blauer Indolyl-Farbstoff entsteht (J. Miller, so.). Aus den isolierten Kolonien wurden mit bekannten Methoden (W. Arber et al., in: Lambda II, R.W. Hendrix et al., Eds., Gold Spring Harbor, New York 1983, S. 433) Phagenlysate hergestellt. Diese Phagen wurden auf den Stamm 783-4 titriert, der eine supF- Mutation zur Suppression der 57-Mutation des Phagen Lambda plac5 und das Episom aus dem Stamm BMH 71-18 (J. Messing et al., Proc. Natl. Acad. Sci. U.S.A. 74 (1977) 3542) aufweist. Das Episom wies die den Laktose-Repressor überproduzierende Mutation iq auf. Auf dem Indikatorstamm 783-4 gewachsene Plaques wurden mit einer 0,5M Catechollösung besprüht. Die Plaques von Phagen, die das xylE-Gen tragen, wurden dadurch gelb gefärbt. Solche Phagen wur den gereinigt.The plasmid pFM24 was transformed into the E. coli strain 50-1 (M. Mieschendahl, Dotorarbeit, University of Cologne, 1981). Strain 50-1 is the E. coli strain GSH50 (J. Miller, Experiments in Molecular Genetics, Gold Spring Harbor, New York 1972), which is lysogenic for the lambda phage Lambda placs (K. Ippen et al., J. Bacteriol. 108, 5, 1971). This phage has parts of the lac operon including the lactose promoter and the lacZ gene. The lac sequences in the plasmid pMM8 are homologous to the sen lac sequences of the phage lambda plac5. The formation of homogeneous (J. Miller, see above) led to the recombination of the lac region of the plasmid pFM24 and the lac region of the phage lambda plac5, as a result of which the xylE gene was transferred from the plasmid to the phage. As a result, the lacZ gene of the phage was inactivated at the same time, so that the sought homogenotes could be isolated as white colonies on x-gal indicator plates. X-gal is a colorless 5-bromo-4-chloro-3-indolyl-beta-galactoside, from which a blue indolyl dye is formed after cleavage by beta-galactosidase, the gene product of the lacz gene (J. Miller, supra .). Phage lysates were prepared from the isolated colonies using known methods (W. Arber et al., In: Lambda II, RW Hendrix et al., Eds., Gold Spring Harbor, New York 1983, p. 433). These phages were titrated to strain 783-4, which contains a supF mutation to suppress the 57 mutation of phage lambda plac5 and the episome from strain BMH 71-18 (J. Messing et al., Proc. Natl. Acad. Sci. USA 74 (1977) 3542). The episome had the mutation i q that overproduced the lactose repressor. Plaques grown on the indicator strain 783-4 were sprayed with a 0.5M catechol solution. As a result, the plaques of phages which carry the xylE gene were stained yellow. Such phages were cleaned.
Die Fig. 1 zeigt die Plasmidkarten der Plasmide pMM8, pEPA53 und pFM24. Fig. 1 shows the plasmid maps of the plasmids pMM8, pEPA53 and pFM24.
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DE4103289A DE4103289A1 (en) | 1991-02-04 | 1991-02-04 | Determn. of lactose repressor over-producing microorganisms - using a phage with a marker gene contg. C-terminal with a lactose operator and promoter which forms an anti-sense RNA of the marker |
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DE4103289A DE4103289A1 (en) | 1991-02-04 | 1991-02-04 | Determn. of lactose repressor over-producing microorganisms - using a phage with a marker gene contg. C-terminal with a lactose operator and promoter which forms an anti-sense RNA of the marker |
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WO1997010360A1 (en) * | 1995-09-13 | 1997-03-20 | Chiron Corporation | Method and construct for screening for inhibitors of transcriptional activation |
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US4861709A (en) * | 1985-05-31 | 1989-08-29 | Technicon Research A.G. | Detection and/or identification of microorganisms in a test sample using bioluminescence or other exogenous genetically-introduced marker |
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US4861709A (en) * | 1985-05-31 | 1989-08-29 | Technicon Research A.G. | Detection and/or identification of microorganisms in a test sample using bioluminescence or other exogenous genetically-introduced marker |
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WO1997010360A1 (en) * | 1995-09-13 | 1997-03-20 | Chiron Corporation | Method and construct for screening for inhibitors of transcriptional activation |
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