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  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
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  • 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
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
  • C12N1/205—Bacterial isolates
• • 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
• • 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
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/185—Escherichia
  • C12R2001/19—Escherichia coli

Definitions

• the invention relates to a plasmid-free clone of E. coli strain DSM 6601, a process for its preparation and the use of the bacteria thus obtained as a cloning vehicle.
• E. coli a naturally occurring bacterium particularly in the human and animal intestines, has long been the subject of detailed microbiological and genetic engineering studies and is used in genetic engineering in particular for the cloning and / or expression of certain genes or proteins.
• strains of the genus Escherichia are pathogenic outside of the intestinal lumen and generally lead to infections in the affected areas.
• An apathogenic strain of the genus Escherichia coli is the strain DSM 6601 deposited with the German Collection of Microorganisms, which deviates in some genetic characteristics from all other E. coli strains.
• this strain is difficult to genetically manipulate only under difficult circumstances and in some cases and thus can not be used as a simple cloning agent.
• E. coli DSM 6601 naturally contains two plasmids, called pMutI or pMut2, having a size of 3177 and 5552 kb, respectively. These plasmids and their DNA sequences are described, for example, in US Pat. No. 6,391,631.
• the object of the invention is to develop a plasmid-free E. coli strain which is otherwise genetically identical to the original strain in terms of its genomic DNA.
• BESTATIGUNGSKOPIE The above object is achieved by providing a plasmid-free clone of the E. coli strain DSM 6601, as well as by a method for producing such a clone.
• Fig. 1 shows in schematic form the procedure for the preparation of the plasmid-free clone of the strain DSM 6601;
• Fig. 2 shows the physical map of the plasmid pMutl-Tc
• Fig. 3 shows physical map of the plasmid pMut2-Kn.
• the plasmids pMut1 and pMut2 naturally occurring in the E. coli strain DSM 6601 are in each case labeled with an antibiotic resistance in a first step according to the method of the invention.
• the plasmids are isolated according to conventional methods and introduced the resistance gene at a desired location.
• the resistance gene is introduced into the respective plasmid together with an expression cassette which contains a promoter which may be constitutive or else inducible.
• the plasmids thus obtained are then introduced into a suitable host according to conventional methods, for example the CaCl 2 method or electroporation, and cloned there, wherein the resistance gene introduced into the respective plasmid is the selection of the J
• a host suitable for cloning the resistance gene carrying plasmid are, for example, the E. coli strains DH5 ⁇ or E. coli HB101.
• the plasmid carrying the resistance gene is isolated and then the sacB gene is introduced into the plasmids to provide another marker.
• the plasmids thus obtained or labeled are then introduced into the E. coli strain DSM 6601.
• the E. coli DSM 6601 are grown on medium containing the antibiotic / antibiotics for which the resistance genes introduced into the plasmids in the previous steps confer resistance.
• the bacteria thus grown are transferred to a nutrient medium which inhibits the growth of bacteria containing the sacB, thereby exerting a selection pressure which essentially allows only the growth of bacteria which have lost the sacB gene.
• a selection pressure which essentially allows only the growth of bacteria which have lost the sacB gene.
• DSM 6601 ⁇ pMutl / 2 have shown that it is a particularly good ⁇ acceptor for foreign DNA, if this is integrated into its own, present in isolated plasmids, ie, the own plasmids as cloning vectors for the Foreign DNA act. Furthermore, since they are derived from an apathogenic strain, they can be used to treat disorders of the gastrointestinal tract in animals and humans.
• adhesins which optionally promote the specific bacteria-specific adhesion of the bacteria to the mucosa of, for example, cattle and / or pigs and thus the hamper or prevent the growth of other pathogenic microorganisms.
• the two naturally occurring plasmids pMutl and pMut2 of the wild-type DSM 6601 were isolated according to the plasmid Midiprep protocol of QIAGEN (QIAGEN Plasmid Purification Handbook 12/2002, pages 16-20).
• the tetracycline resistance cassette derived from the vector pBR322 was selected.
• the associated promoter was taken from the plasmid pASK75.
• the plasmid resulting from this cloning was excised with pKS- tetA tet P / o ⁇ j as insert tetA te ⁇ o (insert size 1.5 kb) was excised with the restriction enzymes XbaI and HindIII.
• the Xb all HindIII-& x & gm & ⁇ X was introduced into the plasmid pMut1 via an NtM restriction site.
• the restriction mixture was purified via a column (Quiagen, PCR Purification Kit) and subjected to Klenow treatment in order to form "blunt ends.”
• a dephosphorylation was carried out Then, the vector pMut1 and the insert tet-4 tetp / o were ligated to transform the E. coli K-12 strain DH5 ⁇ .
• plasmid DNA (1-100 ng) were mixed with 40 ⁇ l of competent cells thawed on ice and incubated on ice for 5 minutes. Thereafter, this batch is pipetted free of air bubbles between the two electrodes of a sterile and precooled 2 mm electroporation cuvette. The cuvette is well dried and inserted into the electrode holder. After carrying out the electric pulse at 2.5 kV, 200 ⁇ and 25 ⁇ F, the bacterial suspension was rinsed out of the cuvette with 1 ml of LB medium and incubated at 37 ° C. for 1-2 hours in a shaker.
• the bacteria were removed by centrifugation and the supernatant was removed to 100 ⁇ l.
• the sediment was resuspended in the remaining 100 ⁇ l, plated on a Tc-containing selection plate and incubated at 37 ° C overnight.
• the success of the cloning was checked by minipreparation of the respective plasmid of individual bacterial clones.
• the plasmid pMutI labeled with the tetracycline cassette was designated pMutI-Tc.
• Fig. 2 the physical map of the plasmid pMutl-Tc is shown. Given is the insertion site (originally a singular N site) for the D ⁇ A fragment, which mediates tetracycline resistance. This D ⁇ A fragment contains also the unique EcoRI sequence suitable for cloning. Furthermore, the binding sites for the primers Muta 5 and Muta 6 are indicated which are suitable for the specific detection of this plasmid by means of PCR.
• a kanamycin resistance cassette was derived, which was derived from the vector pACYC177.
• the resistance cassette (size 1.34 kb) was excised from it with the restriction enzyme StuI and introduced into the plasmid pMut2 via a 5gflI restriction site.
• the restriction mixture was purified via a column (Quiagen, PCR fraction kit) and the plasmid pMut2 was subjected to a Klenow treatment in order to form "blunt ends" for the cloning
• a Klenow treatment in order to form "blunt ends" for the cloning
• dephosphorylation of the restriction enzyme Bgal-linearized and Klenow enzyme-treated plasmid was carried out, followed by ligating the pMut2 vector and the kanamycin cassette to transform the E.
• coli K-12 strain DH5 ⁇ as described in Example 1
• the transformation was plated out on LB-containing LB agar plates and the success of the cloning was checked by minipreparation of plasmid DNA from individual bacterial clones
• the plasmid pMut2 labeled with the kanamycin cassette was designated pMut2-Kn.
• FIG. 3 shows the physical map of the plasmid pMut2-Kn.
• the insertion site originally a -5 / II site
• the singular and as cloning site suitable ifcoRI sequence are shown.
• Muta 7 to Muta 10 identify those regions which are complementary to the sequences of the primers which are suitable for the specific detection of this plasmid by means of PCR.
• Example 3 Introduction of the sacB gene
• the -racB gene coded for a levan saccharase
• the resistance cassettes-labeled plasmids pMutl-Tc and pMut2-Kn in each case the unique EcoRI restriction cleavage site was selected in both plasmids.
• the ⁇ zcB gene (size 2.6 kb) was isolated from the plasmid pCVD442 with the restriction enzyme PstI.
• the success of the cloning was achieved by minipreparation of plasmid DNA of individual bacterial clones and subsequent restriction analysis
• the sacB-Gsn labeled plasmids pMutl-Tc and pMut2-Kn were designated pMutl-TcSac and pMut2-KnSac.
• the plasmid pMutl-TcSac was transformed by electroporation into this strain as described in Example 1. After electroporation to transfer the plasmid pMutl-TcSac into the strain DSM 6601, the batch was plated on Tc-containing (50 ⁇ g / ml) LB plates. The resulting tetracycline-resistant bacterial clones were checked for the possession of the plasmid pMutl-TcSac and the associated loss of the naturally occurring plasmid pMutl.
• the loss of the plasmid pMutl was detected after preparation of the plasmid DNA and restriction digestion of the same with the enzyme EcoRI after electrophoretic separation of the linearized plasmids by the absence of the pMutl representing DNA band.
• LB medium consists of 10 g of peptone from casein, 5 g of yeast extract and 5 g of sodium chloride to 1 1 distilled Water.
• Sucrose-containing LB medium was prepared by adding to the autoclaved medium after cooling to 45 ° C from a sterile filtered 50% (w / v) sucrose stock solution to a final concentration of 10%.) The plates were incubated at 30 ° C. Under these conditions, only those clones that no longer express sacB, ie those that have lost the A2cB gene-bearing plasmid, can replicate. The resulting strain DSM 6601 ⁇ pMutl was checked for the loss of the plasmid pMutl-TcSac.
• the plasmid pMut2-KnSac was then introduced into strain DSM 6601 ⁇ pMut1 by electroporation.
• the batch was plated on Kn containing (50 ⁇ g / ml) LB plates.
• the obtained kanamycin-resistant bacterial clones were checked for the possession of the plasmid pMut2-KnSac and the associated loss of the naturally occurring plasmid pMut2.
• one of these clones was grown overnight in LB medium with 10% sucrose at 30 ° C and then plated on LB plates with 10% sucrose. The plates were incubated at 30 ° C.
• the resulting strain DSM 6601 ⁇ pMutl / 2 was checked for the loss of the plasmid pMut2-KnSac.
• pulsed-field gel electrophoresis was performed on the plasmid-free strain DSM 6601 ⁇ pMutl / 2 in order to exclude any chromosomal alterations of the strain. It was found that no changes were detectable and further investigations have shown that the plasmid-free clone shows no morphological, biochemical or fermentative changes.

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