WO1999025869A1 - Dna-sequenzen aus fimbriengenen des escherichia coli-stamms dsm 6601 - Google Patents
Dna-sequenzen aus fimbriengenen des escherichia coli-stamms dsm 6601 Download PDFInfo
- Publication number
- WO1999025869A1 WO1999025869A1 PCT/EP1998/007397 EP9807397W WO9925869A1 WO 1999025869 A1 WO1999025869 A1 WO 1999025869A1 EP 9807397 W EP9807397 W EP 9807397W WO 9925869 A1 WO9925869 A1 WO 9925869A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- dna sequences
- dna
- escherichia coli
- genes
- fimbriae
- Prior art date
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Classifications
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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
-
- 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
- C12N15/70—Vectors or expression systems specially adapted for E. coli
Definitions
- the invention relates to DNA sequences from the Fimbrien gene clusters of Escherichia coli strain DSM 6601.
- Escherichia coli is a gram-negative bacterium that is found in the human and animal intestinal flora, but also extraintestinal. E. coli occurs in numerous variants which differ in terms of capsule antigens, surface antigens and flagella antigens and can therefore be divided into numerous serological types. The classification according to the serotypes, however, does not allow any statement about the different virulence of the germs. Representatives of one and the same serotype can have a different pathogenicity potential in both the human and animal body, which in extreme cases can range from avirulent to highly pathogenic.
- the E. coli strain DSM 6601 belongs to the serogroup 06: K5 and is rated as non-human or animal pathogenic.
- This strain has two chromosomally coded fimbrial gene clusters, namely a type I (fim) or F1 C (focj gene cluster. It is known that the fimbriae of this strain carry an adhesin. Adhesins are structures which are involved in the attachment of the bacterial Organism to other cells play an important role.
- Fimbrial genes are mainly used in analysis and diagnostics. Applications are also available in medicine and nutritional physiology or in biotechnology. Fimbrial genes or their main subunits can thus be used, for example, to characterize a particular strain, so that there is a need for further studies on the sequence of these genes.
- Steps 2-4 were repeated 30 times.
- focA1 5 '-CTC ACA TTG CAT TTA TGA AG-3' focA2: 5'-GGT ATA TAT CCG TTA CAC TG-3 '
- Step 1 denature for 3 min at 95 ° C
- Step 2 45 sec 95 ° C
- Step 3 45 sec 51 ° C
- Step 4 45 sec 72 ° C
- Step 5 5 min at 72 ° C
- PCR products obtained in Examples 1 and 2 are based on the method of Sambrook et al. (Sambrook, J., Fritseh, EF, and Maniatis, T., Molecular Cloning: A laboratory manual, Cold Spring Harbor Laboratory, second edition (1989), Cloning, Transformation: 1 .53 - 1 .84; PCR: 14.00 - 14.35) was cloned into the vector pUC 18 and the resulting plasmid DNA was transformed into the E. coli K12 strain DH5.
- the plasmid DNA is isolated based on the method of Bimboim et al. (Birnboim, A.C. and Doly, J. (1979) Nucl. Acids Res. 7: 1 51 3-1523. A rapid alkaline extraction procedure for screening recombinant plasmid DNA).
- 3 ml LB medium are inoculated with a bacterial colony and shaken overnight at 37 ° C.
- This culture is centrifuged off in an Eppendorf tube, the media residue is removed with a pipette.
- the cell sediment is resuspended with 100 ⁇ ⁇ solution I (50 mM glucose; 10 mM EDTA, pH 8; 25 mM Tris-HCl, pH 8).
- 100 ⁇ ⁇ solution I 50 mM glucose; 10 mM EDTA, pH 8; 25 mM Tris-HCl, pH 8
- 200 ⁇ l of solution II 0.2 N NaOH; 1% SDS
- the mixture is mixed until clear and the Eppendorf tube is left on ice for a further 5 minutes.
- the plasmid DNA is precipitated with 2 volumes of ice-cold ethanol and centrifuged for 10 minutes. The pellet is washed with 70% ethanol and dried in the Speedvac. The plasmid DNA is resuspended in 20 ⁇ ⁇ H 2 O bidest and stored at -20 ° C.
- DNA sequencing was based on the method of Sanger et al. (Sanger, F., Nicklen, S. and Coulson, A.R. (1977) Proc. Natl. Acad. Sci. USA 74: 5463-5467. DNA sequencing with chain terminating inhibitors).
- DNA sequencing was carried out using the T7 sequencing kit from Phamacia-LKB.
- the DNA is double-distilled water and with 3 ⁇ ⁇ 3 M Na-acetate, pH 4.8 and 7 ul H 2 O 60 ⁇ ⁇ ice-cold ethanol abso
- the precipitated DNA is centrifuged off for 10 min, washed with 70% ethanol and dried.
- the denatured DNA is suspended in 10 ⁇ ⁇ H 2 O bidest and mixed with 2 ⁇ ⁇ annealing buffer and 2 ⁇ l primer (40 ng) mixes. The mixture is incubated at 37 ° C. for 20 min so that the primer can bind to the template DNA. The reaction mixture is cooled for 10 min at room temperature and then either used immediately for the labeling reaction or frozen at -20 ° C.
- DNA sequences can also be synthetically produced in a manner known per se and certain sequence sections can be used as primers, which then enables the identification of E. coli strain DSM 6601 without any problems. But there is also the possibility of the corresponding DNA sequences of this strain are to be introduced into other E. coli strains by genetic engineering, in order, for example, to modify the behavior when cells are attached and thus to influence the colonization properties of other strains.
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Plant Pathology (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Saccharide Compounds (AREA)
Abstract
Description
Claims
Priority Applications (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EEP200000265A EE200000265A (et) | 1997-11-19 | 1998-11-18 | Escherichia coli tüve DSM 6601 fimbriageenidest pärinevad DNA järjestused ja nende kasutamine |
PL98340329A PL340329A1 (en) | 1997-11-19 | 1998-11-18 | Dna sequence of genes encoding fimbriae of escherichia coli dsm 6601 strain |
JP2000521232A JP2001523470A (ja) | 1997-11-19 | 1998-11-18 | エシェリキア・コリ菌株dsm6601のフィムブリエ遺伝子からのdna配列 |
EP98962355A EP1032711A1 (de) | 1997-11-19 | 1998-11-18 | Dna-sequenzen aus fimbriengenen des escherichia coli-stamms dsm 6601 |
HU0100337A HUP0100337A3 (en) | 1997-11-19 | 1998-11-18 | Dna sequences of genes from fimbriae d'escherichia coli strain dsm 6601 |
US09/554,834 US6376185B1 (en) | 1997-11-19 | 1998-11-18 | DNA sequences of genes from fimbriae d'escherichia coli strain DSM 6601 |
NO20002549A NO20002549L (no) | 1997-11-19 | 2000-05-18 | DNA-sekvenser av fimbriagener av Escherichia coli stamme DSM 6601 |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE19751242A DE19751242C2 (de) | 1997-11-19 | 1997-11-19 | DNA-Sequenzen aus Fimbriengenen von Escherichia coli Stamm DSM 6601 |
DE19751242.9 | 1997-11-19 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1999025869A1 true WO1999025869A1 (de) | 1999-05-27 |
Family
ID=7849196
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/EP1998/007397 WO1999025869A1 (de) | 1997-11-19 | 1998-11-18 | Dna-sequenzen aus fimbriengenen des escherichia coli-stamms dsm 6601 |
Country Status (9)
Country | Link |
---|---|
US (1) | US6376185B1 (de) |
EP (1) | EP1032711A1 (de) |
JP (1) | JP2001523470A (de) |
DE (1) | DE19751242C2 (de) |
EE (1) | EE200000265A (de) |
HU (1) | HUP0100337A3 (de) |
NO (1) | NO20002549L (de) |
PL (1) | PL340329A1 (de) |
WO (1) | WO1999025869A1 (de) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE19915772C2 (de) * | 1998-11-18 | 2001-08-30 | Pharma Zentrale Gmbh | Verfahren zur Identifizierung von Escherichia coli Stamm DSM 6601 |
US6376185B1 (en) | 1997-11-19 | 2002-04-23 | Pharma-Zentrale Gmbh | DNA sequences of genes from fimbriae d'escherichia coli strain DSM 6601 |
US6489107B1 (en) | 1997-11-19 | 2002-12-03 | Pharma-Zentrale Gmbh | Method for identifying Escherichia coli strain DSM 6601 |
US7611711B2 (en) | 2001-01-17 | 2009-11-03 | Vegenics Limited | VEGFR-3 inhibitor materials and methods |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998044134A2 (de) * | 1997-04-02 | 1998-10-08 | Pharma-Zentrale Gmbh | Bakterielle plasmide |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE19751242C2 (de) | 1997-11-19 | 2001-02-08 | Pharma Zentrale Gmbh | DNA-Sequenzen aus Fimbriengenen von Escherichia coli Stamm DSM 6601 |
-
1997
- 1997-11-19 DE DE19751242A patent/DE19751242C2/de not_active Expired - Lifetime
-
1998
- 1998-11-18 PL PL98340329A patent/PL340329A1/xx unknown
- 1998-11-18 EP EP98962355A patent/EP1032711A1/de not_active Withdrawn
- 1998-11-18 EE EEP200000265A patent/EE200000265A/xx unknown
- 1998-11-18 HU HU0100337A patent/HUP0100337A3/hu unknown
- 1998-11-18 JP JP2000521232A patent/JP2001523470A/ja active Pending
- 1998-11-18 US US09/554,834 patent/US6376185B1/en not_active Expired - Fee Related
- 1998-11-18 WO PCT/EP1998/007397 patent/WO1999025869A1/de not_active Application Discontinuation
-
2000
- 2000-05-18 NO NO20002549A patent/NO20002549L/no not_active Application Discontinuation
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998044134A2 (de) * | 1997-04-02 | 1998-10-08 | Pharma-Zentrale Gmbh | Bakterielle plasmide |
Non-Patent Citations (5)
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6376185B1 (en) | 1997-11-19 | 2002-04-23 | Pharma-Zentrale Gmbh | DNA sequences of genes from fimbriae d'escherichia coli strain DSM 6601 |
US6489107B1 (en) | 1997-11-19 | 2002-12-03 | Pharma-Zentrale Gmbh | Method for identifying Escherichia coli strain DSM 6601 |
DE19915772C2 (de) * | 1998-11-18 | 2001-08-30 | Pharma Zentrale Gmbh | Verfahren zur Identifizierung von Escherichia coli Stamm DSM 6601 |
US7611711B2 (en) | 2001-01-17 | 2009-11-03 | Vegenics Limited | VEGFR-3 inhibitor materials and methods |
Also Published As
Publication number | Publication date |
---|---|
HUP0100337A3 (en) | 2005-08-29 |
DE19751242A1 (de) | 1999-05-27 |
US6376185B1 (en) | 2002-04-23 |
HUP0100337A2 (hu) | 2001-06-28 |
NO20002549L (no) | 2000-07-18 |
NO20002549D0 (no) | 2000-05-18 |
EE200000265A (et) | 2001-06-15 |
DE19751242C2 (de) | 2001-02-08 |
EP1032711A1 (de) | 2000-09-06 |
PL340329A1 (en) | 2001-01-29 |
JP2001523470A (ja) | 2001-11-27 |
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