Title of Invention	"PLASMID-FREE CLONE OF E. COLI STRAIN DSM 6601"
Abstract	A plasmid-free clone of Escherichia coli strain DSM 6601, wherein said clone lacks the naturally occurring two plasmids pMUTl and pMUT2 and the method of preparing the same.

Full Text

The invention relates to a plasmid-free clone of E. coli strain DSM 6601, a method of its preparation and the use of the bacteria obtained in this manner as a cloning vehicle. E. coli, a bacteria that occurs in nature, especially in the intestines of humans and animals, has long been the subject of intensive microbiological and genetic engineering research and is used in genetic engineering in particular for cloning and/or expressing certain genes and/or proteins. Most strains of the genus Escherichia are pathogenic outside of the lumen of the bowels and generally cause infections at the affected sites. A non-pathogenic strain of the genus Escherichia coli is the strain DSM 6601 deposited in the German Collection for Microorganisms, that deviates in a few genetic features from all other E. coli strains. However, it turned out that this strain can be manipulated genetically only under difficult circumstances and partially not at all and therefore cannot be used as a simple cloning means. E. coli DSM 6601 naturally contains two plasmids designated as pMutl or pMut2 that have a size of 3177 and 5552 kb. These plasmids and their DNA sequences are described, e.g., in US 6, 391, 631. Starting with the consideration that the pathogenicity and non-pathogenicity of E. coli bacteria are obviously partially controlled by their plasmids, and with the consideration that the partially occurring "genetic resistance" of E. coli strains might also be connected to its cryptic plasmids, the invention has the basic problem of developing a plasmid-free E. coli strain that is otherwise genetically totally identical to the additional strain as regards its genomic DNA. The above problem is solved by making available a plasmid-free clone of E. coli strain DSM 6601 and by a method of preparing such a clone. Figure 1 shows a schematic of the method for preparing the plasmid-free clone of strain DSM 6601. Figure 2 shows the physical map of plasmid pMutl-Tc. Figure 3 shows the physical map of plasmid pMut2-Kn. It turned out in the exhaustive investigations that led to the present invention that plasmid-free clones of strain DSM 6601 cannot be prepared at all with normal genetic engineering methods or can be prepared only with great difficulty so that special paths must be taken in order to generate such clones. Since the wild type of the strain has two plasmids of different sizes in addition to its genomic DNA, the elimination of these plasmids must take place in several steps that take place in part in parallel. In order to prepare the clones in accordance with the invention the plasmids pMutl and pMut2 occurring naturally in E. coli strain DSM 6601 are marked in accordance with the method of the invention in a first step with an antibiotic resistance. To this end the plasmids are isolated according to traditional methods and the resistance gene inserted at the desired site. According to a preferred embodiment the resistance gene is inserted into the particular plasmid together with an expression cassette containing a promoter that can be constitutive or also inducible. Plasmids obtained in this manner are then introduced into a suitable host according to traditional methods, e.g., the CaCl2 method or electroporation where they are cloned, during which the resistance gene inserted into the particular plasmid facilitates the selection of the host cells carrying the plasmid. A host suitable for cloning the plasmid carrying a resistance gene is, e.g., the E. coli strain DH5a or E. Coli HB101. The plasmids carrying the resistance gene are isolated and the sacB gene is subsequently introduced into the plasmids in order to make another marker available. The plasmids obtained and marked in this manner are then introduced into E. coli strain DSM 6601. After transformation the E, coli DSM 6601 are cultivated on a medium containing the antibiotic/antibiotics for which the resistance genes introduced in the preceding steps into the plasmids impart resistance. By cultivating in such a medium, only clones grow that are resistant to the antibiotics contained in the nutrient medium. Furthermore, the bacteria lose excess genetic material since they no longer require the original plasmids pMutl and pMut2 for their growth so that the bacteria finally contain only the modified plasmas pMutl and pMut2 (that contain the resistance gene/genes and the sacB gene). In a further step the bacteria cultivated in this manner are transferred into a nutrient medium that inhibits the growth of bacteria containing the sacB, during which a selection pressure is exerted that substantially only permits the growth of bacteria that have lost the plasmid carrying the sacB gene. This can take place by cultivating the strain at 30°C in the presence of 10% saccharose, since under such conditions only those clones can replicate that have lost the plasmid carrying the sacB gene. As a consequence, a plasmid-free derivative of strain DSM 6601 is obtained. It was now found that the clones in accordance with the invention, with the condition of the loss of the plasmids, did not experience any change of the genomic DNA and can surprisingly be readily used as cloning vehicles. Thus, they can be safely used in the laboratory as host cell for the cloning and expression of a plurality of genes and proteins. Experiments with strain DSM 6601 ApMutl/2 have shown that it is an especially good accepter for foreign DNA when the latter is integrated into its own plasmids present in isolated form, that is, therefore its own plasmids function as cloning vectors for the foreign DNA. Furthermore, since they are derived from a non-pathogenic strain, they can be used for the treatment of disturbances of the gastrointestinal tract in animals and humans. To this end they can be transformed, if desired with foreign genes that further the adhesion of the bacteria to the rnucosa such as, e.g., adhesines that further the adhesion of the bacteria, optionally host-animal specifically, to the mucosa of, e.g., cattle and/or swine and thus hinder or prevent the growth of other pathogenic microorganisms. The present invention will now be explained in detail with reference made to the examples. Example 1 Modification of pMut 1 The two naturally occurring plasmids pMutl and pMut2 of wild type isolated in accordance with the plasmid midi-prep protocol of QIAGEN (QIAGEN Plasmid Purification Handbook 12/2002, pages 16-20). The tetracycline resistance cassette derived from vector pBR322 was selected for marking the plasmid. The associated promoter was taken from plasmid pASK75. The plasmid resulting from this cloning was designated by pKS-tetAtetp/0. The insert tetAtetp/0 (insert size 1 .5 kb) was cut out with restriction enzymes Xbal and Hwdlll. The Xfozl /Hindlll fragment was introduced into plasmid pMutl via a Ndel restriction site. To this end the restriction batch was purified via a column (Qulagen, PCR purification kit) after restriction digestion of the plasmids with the appropriate enzymes and subjected to a Klenow treatment in order to fonn blunt ends. In order to prevent a religation of the plasmid pMutl a dephosphorylation of the plasmid linearized with restriction enzyme Ndel and treated with Klenow enzyme was carried out. Subsequently, the vector pMutl and the insert tetAtetp/0 were litigated and the E. coli K-12 strain DH5a transformed therewith. In order to prepare competent cells 150 mm LB medium (Lurea-Bertani medium) was inoculated with 1.5 mm of a UN [? - non-standard abbreviation - overnight? - see end of paragraph] culture and agitated at 37°C until an OD6oo-0-5. The bacterial culture was then incubated 20 minutes on ice and centrifuged 10 minutes with 4000 rpm at 4°C. The bacterial pellet was washed 3 times in sterile, ice-cold 10% glycerol, at first with 100%, then with 50% and finally with 10% of the initial volume. Finally, the washed pellet was re-suspended in 300 ul 10% glycerol, aliquoted (40 ju.1) and stored at -80°C. For the transformation of bacterial cells 1-2 u.1 plasmid DNA (1-100 ng) was mixed with 40 ul competent cells thawed on ice and incubated 5 minutes on ice. This batch was then pipetted free of air bubbles between the two electrodes of a sterile and pre-cooled 2 mm electroporation cuvette. The cuvette was dried off well and inserted into the electrode holder. After the electrical impulse had been performed at 2.5 kV, 200 H and 25 uF the bacterial suspension was washed out of the cuvette with 1 mm LB medium and incubated at 37°C for 1-2 hours in an agitator. The bacteria were subsequently centrifuged off and the supernatant drawn off up to 300 jil. The sediment was re-suspended in the remaining 100 ul, plated out onto a Tc-containing selection plate and incubated UN [overnight?] at 37°C. The success of the cloning was tested by minipreparation of the particular plasmid of individual bacterial clones. The plasmid pMutl marked with the tetracycline cassette was designated as pMutl-Tc. Figure 2 shows the physical map of plasmid pMutl-Tc. It indicates the insertion site (originally a singular Ndel site) for the DNA fragment that imparts tetracycline resistance. This DNA fragment also contains the singular Eco-RJ sequence suitable for clonings. Furthermore, the binding sites for the primers muta 5 and muta 6 are indicated that are suitable for the specific demonstration of this plasmid by PCR. Example 2 Modification of pMut2 A kanamycin resistance cassette was selected for the marking of plasmid pMut2 that was derived from vector pACYC177. To this end the resistance cassette (size 1 ,34 kb) was cut out of the latter with the restriction enzyme Stul and introduced via a BglU restriction cleavage site into plasmid pMut2, After restriction digestion of the plasmids with the appropriate enzymes the restriction batch was purified via a column (Quiagen PCR fraction kit) and plasmid pMut2 subjected to a Klenow treatment in order to form blunt ends for the cloning. In order to avoid a religation of plasmid pMut2 a dephosphorylation linearized with restriction enzyme Bg/II and treated with Klenow enzyme was carried out. Subsequently, vector pMut2 and the kanamycin cassette were litigated and the E. coli K-12 strain DH5a transformed as described in example 1. The transformation was plated out onto Kn-containing LB agar plates. The success of the cloning was tested by minipreparation of plasmid DNA of individual bacterial clones. Plasmid pMut2 marked with the kanamycin cassette was designated as pMut2-Kn. Figure 3 shows the physical map of plasma pMut2-Kn. The insertion site (originally a BglU site) for the kanamycin resistance cassette as well as the singular Eco-Rl sequence suitable as cloning site are sketched in. Muta 7 to Muta 10 characterize those areas complementary to the sequences of the primers that are suitable for the specific demonstration of this plasmid by PCR. Example 3 Introduction of the sacB gene In order to introduce the sacB gene (coded for a Levan saccharose) into the plasmids pMutl-Tc and pMut2-Kn, the particular singular Eco-Rl restriction cleavage site was selected in both plasmids. The sacB gene (size 2 .6 kb) was isolated from the plasmid pCVD442 with the restriction enzyme Pstl. In order to prepare the vectors pMutl-Tc and pMut2-Kn, these plasmids were linearized with Eco-Rl and subsequently submitted to a Klenow treatment and dephosphorylated in order to form blunt ends. The insert sacB was purified with Pstl after the restriction digestion and treated with Klenow enzyme. The linearized vectors and the insert were ligated and the E. coli K-12 strain DH5a, that was rendered competent as described in example 1, was transformed therewith. The success of the cloning was checked by minipreparation of plasmid DNA of individual bacterial clones and subsequent restriction analysis. The plasmids pMutl-Tc and pMut2-Kn marked with the sacB gene were designated as pMutl-TcSac and pMut2-KnSac, Example 4 Preparation of a plasmid-free clone of E. coli strain DSM 6601 At first the plasmid pMutl-TcSac was transformed by electroporation into this strain as described in example 1. After the electroporation in order to transfer plasmid pMutl-TcSac into strain DSM 6601 the batch was plated out onto LB plates containing Tc (50 u.g/ml). The tetracycline-resistant bacterial clones obtained were checked for the possession of plasmid pMutl -TcSac and the associated loss of the naturally occurring plasmid pMutl. The loss of plasmid pMutl was demonstrated after preparation of the plasmid DNA and restriction digestion of the same with the enzyme Eco-R3 after electrophoretic separation of the linearized plasmids by the lack of the DNA bands representing pMutl. Subsequently, one of these clones was attracted overnight in LB medium with 10% saccharose at 30°C and then plated out onto LB plates with 10% saccharose (LB medium consists of 10 g peptone from casein, 5 g yeast extract and 5 g sodium chloride per 1 1 distilled water). Saccharose-containing LB medium was prepared in that a part of a sterilely filtered, 50% (wt./vol.) saccharose stock solution was added to the autoclaved medium after it had cooled off to 45°C up to an end concentration of 10%). The plates were incubated at 30°C. Under these conditions only those clones can replicate that no longer express sacB, that is, they have lost the plasmid carrying the sacB gene. The resulting strain DSM 6601ApMutl was checked for the loss of plasmid pMutl-TcSac. Then, plasmid pMut2-KnSac was introduced by electroporation into strain DSM 6601ApMutl. After the electroporation in order to transfer plasmid pMutZ-TcSac into strain DSM 6601ApMutl, the batch was plated out onto LB plates containing Kn (50 u.g/ml). The kanamycin-resistant bacterial clones obtained were checked for the possession of plasmid pMut2-KnSac and the associated loss of the naturally occurring plasmid pMut2, Subsequently, one of these clones was attracted overnight in LB medium with 10% saccharose at 30°C and then plated out onto LB plates with 10% saccharose. The plates were incubated at 30°C. The resulting strain DSM 6601 ApMutl/2 was checked for the loss of plasmid pMut2-KnSac. Furthermore, a Pulsfeld gel electrophoresis of the plasmid-free strain DSM 6601 ApMutl/2 was carried out in order to exclude any chromosomal modifications of the strain. It was determined that no changes could be demonstrated and further examinations showed that the plasmid-free clone displayed no morphological, biochemical or fermentative changes. WE CLAIM; 1. A plasmid-free clone of Escherichia coli strain DSM 6601, wherein said clone lacks the naturally occurring two plasmids pMUTl and pMUT2. 2. The method of preparing a plasmid-free clone as claimed in Claim 1, characterized by the following steps: a) The introduction of a resistance gene of the kind such as herein described into plasmids pMutl and pMut2, b) The introduction of the sacB gene into the plasmids obtained in step a), c) The introduction of the plasmids obtained in step b) into the E. coli strain DSM 6601 and cultivation of the strain under conditions as herein described in which the naturally occurring plasmids pMutl and pMut2 are displaced by the plasmids obtained in step b); and d) Cultivation in a manner as herein described of the clones obtained in step c) that substantially only permit the growth of bacteria that lack the sacB gene. 3. The method as claimed in Claim 2, wherein the resistance genes are present in an expression cassette. 4. The method as claimed in Claim 2 or 3, wherein the resistance genes are selected under tetracycline resistance or kanamycin resistance. 5. The method as claimed in any one of Claims 2 to 4, wherein that plasmid pMutl is marked with a tetracycline resistance cassette and the sacB gene and that the original plasmid pMut2 is marked with a kanamycin resistance cassette and the sacB gene. 6. The method as claimed in any one of Claims 2 to 5, in which the bacteria transformed with plasmid pMutl, that is marked with a tetracycline resistance cassette and the sacB gene, are cultivated on plates containing tetracycline and subsequently on plates containing saccharose, and that after the elimination of plasmid pMutl in the first step the elimination of plasmid pMut2 takes place by cultivation on kanamycin plates and further cultivation on saccharose plates.

Documents:

1087-DELNP-2005-Abstract-(23-10-2008).pdf

1087-delnp-2005-abstract.pdf

1087-DELNP-2005-Claims-(04-11-2008).pdf

1087-DELNP-2005-Claims-(23-10-2008).pdf

1087-delnp-2005-claims.pdf

1087-delnp-2005-complete specification (granted).pdf

1087-DELNP-2005-Correspondence-Others-(23-10-2008).pdf

1087-delnp-2005-correspondence-others.pdf

1087-delnp-2005-description (complete).pdf

1087-DELNP-2005-Drawings-(23-10-2008).pdf

1087-delnp-2005-drawings.pdf

1087-DELNP-2005-Form-1-(23-10-2008).pdf

1087-delnp-2005-form-1.pdf

1087-delnp-2005-form-18.pdf

1087-DELNP-2005-Form-2-(23-10-2008).pdf

1087-delnp-2005-form-2.pdf

1087-DELNP-2005-Form-3-(23-10-2008).pdf

1087-delnp-2005-form-3.pdf

1087-delnp-2005-form-5.pdf

1087-DELNP-2005-GPA-(23-10-2008).pdf

1087-delnp-2005-gpa.pdf

1087-DELNP-2005-Others-Document-(23-10-2008).pdf

1087-delnp-2005-pct-search report.pdf

1087-DELNP-2005-Petition-137-(23-10-2008).pdf

1087-DELNP-2005-Petition-138-(23-10-2008).pdf