DE102007048134A1 - Cloning, integration and expression of a gene cluster comprises using gene cassettes comprising sequences that mediate the transfer, integration and expression of a flanked nucleic acid - Google Patents

Abstract

Cloning, integration and expression of a gene cluster comprises preparing gene cassettes comprising sequences that mediate the transfer and integration of a flanked nucleic acid and the expression of all genes in the flanked nucleic acid, producing a recombinant transposon by linking the gene cassettes upstream and downstream of the gene cluster, introducing the transposon into a cell so that the transposon automatically integrates into the cell's genome, and expressing all of the genes of the cluster. Independent claims are also included for: (1) gene cassette for cloning a gene cluster, comprising a promoter sequence and a transposition element; (2) vector for cloning, integration and expression of a gene cluster, comprising at least one gene cassette as above; (3) cell containing a gene cassette or vector as above; (4) culture of a cell as above.

Description

The The invention relates to a process for cloning, integration and Expression of several genes of at least one gene cluster and at least a gene cassette for carrying out the method.

Background of the invention

Microorganisms produce a large number of scientifically, biotechnologically and medically important substances. Almost every day, genome mining or complex metagenome and screening techniques are used to identify new genes responsible for the synthesis of unusual metabolic products with biomedical potential. It turns out that for the synthesis of such metabolic products (usually secondary metabolites) often a larger number of different enzymes is needed, which together form an enzyme complex or metabolic pathway. Interestingly, the genes encoding such enzyme systems, and thus functionally coupled to each other, are mostly organized together in large functional clusters on a chromosome. Among the best known examples of biotechnologically and medically relevant compounds produced by complex enzyme systems are the so-called polyketides and non-ribosomal peptides. They are synthesized by polyketide synthases (PKS) and non-ribosomal peptide synthases (NRPS) in multimodular assembly steps ( Wenzel & Müller, 2005 ) whose genes are localized in large clusters of at least 40 kb in length. However, many of the new bioactive compounds are derived from unicellular organisms that have very slow growth or are not culturable at all (such as symbionts of marine sponges or corals). Thus, the heterologous synthesis of such compounds in established bacterial production strains is often the only way to provide a new metabolite in large quantities.

Around however, a heterologous synthesis of secondary metabolites in a foreign organism basically two prerequisites are met. For one, the entire gene cluster must be isolated from the original organism in be transferred to the bacterial production strain and on the other hand, in the microbial production strain the genetic conditions are created to the alien Being able to express genes functionally. The associated Problems and the solutions described in the literature are explained below with some examples.

1.1 Heterologous Expression of Enzyme Complexes and biosynthetic pathways

The functional expression of an enzyme in a biotechnologically good manageable microorganism such. B. Escherichia coli is in the Usually limited by different factors:

The codon usage

Different microorganisms have very different preferences in their codon usage. If the codon usage between the original and the host organism deviates too much, the translation of the heterologous target genes can be significantly impaired ( Gustaffson et al., 2004 ).

Host-specific DNA elements

Frequently, foreign genetic elements, such as promoters or regulatory regions in the host organism are not recognized or only inadequately recognized, whereby the transcription of the target genes is impaired or can not be initiated ( Lambalot et al., 1996 ).

Biosynthetic background

Both the expression of host-foreign biocatalysts and the synthesis of new metabolites (eg secondary metabolites) provide e.g. T. very specific requirements for the metabolic background of the host organism. So z. For example, for the heterologous synthesis of proteins that require cofactors or prosthetic groups (such as redox enzymes), these cofactors are provided in the expression host in sufficient quantities and incorporated into the host protein ( Drepper et al., 2005 ) or all cofactor synthesis genes of the parent organism are coexpressed in the heterologous system. Similarly, with proteins that have a large misfolding potential; Here functional expression requires a powerful protein folding machinery (so-called chaperones) ( de Marco et al., 2007 ). In addition, there are special requirements for the metabolic activity of the host cell: on the one hand, this plays an important role if an unusual prepress needs to be provided for the production of a connection. On the other hand, however, it was also possible to observe that, depending on the individual metabolic activity of the microbial production strain, completely new compounds with high biotechnological potential could be produced ( Wenzel et al., 2005 ).

In In recent years, various methods have been developed to presented problems in heterologous expression more complex Systematically bypass enzyme systems. All are described Methods basically based on two different principles:

1) Heterologous gene expression in related host organisms

If there is a close affinity between the original organism and the selected production strain, codon usage and promoter structures are often similar, so that the heterologous genes can be expressed quite simply. So z. B. the Dpt gene cluster from Streptomyces roseosporus, which codes for the antibiotic daptomycin, are successfully expressed in Streptomyces lividans ( Miao et al., 2005 ). The disadvantage of this method, however, is basically that a related and well manageable expression strain must be available for the functional expression of a complex enzyme system. In addition, the expression of the target genes is not or only poorly controllable and usually leads only to low protein and product yields.

2) Heterologous gene expression with established expression systems

The expression of many genes of a gene cluster can alternatively be carried out in well-established expression systems ( Kao et al., 1994 ). Although the successive and targeted recloning of clustered genes into corresponding expression plasmids is very time-consuming, this method offers a decisive advantage: The heterologous expression of the cluster genes can thus also be carried out in unrelated expression hosts, since the target genes in the expression vectors used are under the control of host-specific promoters can be made. This also usually allows controlled overproduction of the desired biocatalysts or metabolites in the recombinant microbial production strain.

With this method z. For example, the siderophore Yersiniabactin from Yersinia pestis is successfully produced in E. coli ( Pfeifer et al., 2001 ; Pfeifer et al., 2003 ). For the synthesis of yersiniabactin, a 22 kb gene cluster had to be transferred into the E. coli host strain. Epothilone D, a natural polyketide from Sorangium cellulosum, whose synthesis gene cluster comprises 56 kb, could also be expressed heterologously in Myxococcus xanthum ( Julien & Shah, 2002 ; Lau et al., 2002 ).

1.2 Construction of recombinant plasmids for the expression of clustered genes by genetic engineering

Functional coupled genes of a large cluster are usually not in the same direction of transcription and usually have more as a promoter and a transcription terminator. A such gene arrangement therefore makes the successive incorporation of the individual Genes into corresponding expression vectors for the combined Transcription inevitable. Basically however, commercially available expression systems only for the cloning and expression of a gene or a designed from a few genes transcription unit. Around to produce suitable recombinant expression vectors are described in in most cases, the initial vectors and the target genes-carrying DNA fragments using defined type II restriction endonucleases hydrolyzed and then the compatible ends of the thereby resulting DNA fragments with DNA ligases linked together. The size of the manageable DNA fragments is included however, due to the properties of the restriction endonucleases limited to approx. 10 kb: Restriction enzymes recognize specifically a palindromic nucleotide sequence of six bases, resulting in statistical any DNA fragment of any sequence for each Restriction endonuclease after about 4000 bp a recognition site having. Thus, the recloning of large DNA fragments in one step with conventional genetic engineering methods not possible.

In The literature has so far become two basic methods described to clone various genes around them subsequently to express in the appropriate host:

1) Complex sequence of forced cloning

With First, the individual genes are to be aided by PCR amplified gene cluster amplified. Through the use of suitable Primers that in addition to the homologous sequences also recognition sites for restriction endonucleases the individual sequences are cloned into vectors after the PCR. In further cloning steps, the individual components of the cluster successively merged again.

However, due to the numerous cloning steps and the required complex cloning strategy, this method is very time-consuming ( Wenzel et al., 2004 ). Another disadvantage of this method is that due to the many PCRs, mutations can be incorporated into the genes.

2) Red / ET cloning technology

The Red / ET cloning technology method is based on the principle of homologous recombination ( Zhang et al., 1998 ), which can be carried out in vitro using the isolated proteins RecE and RecT or the homologous proteins Red⎕ and Red⎕. Using this method, a gene cluster from the myxobacterium Stigmatella aurantiaca, which codes for myxochromide S, could be cloned and subsequently expressed heterologously in Pseudomonas putida ( Wenzel et al., 2005 ). In front of the complete mch gene cluster, which codes for myxochromide S, an origin of transfer (oriT) was inserted by homologous recombination, which enabled the transfer of the gene cluster to P. putida. In addition, a host-specific promoter was integrated, which was recognized in contrast to the natural promoter of the gene cluster of P. putida.

Even though by the described methods in the individual case the production of a microbial production strain basically possible is, some problems remain great, especially in the expression Gene clusters exist. So are big gene regions, as already often mentioned by transcription terminators interrupted and the integration of a host-specific promoter After each Transkriptionstermination is technically very complicated. The genes of a cluster are also often not in one orientation arranged, so far more cloning steps necessary are to arrange the genes in an orientation and complete To achieve transcription of all genes of a cluster. Come in addition, that the establishment of a gene cluster on an extrachromosomal DNA element, such as a plasmid, cosmid or BAC, in a heterologous Host is usually not stable.

Summary of the invention

It It is an object of the invention to provide a method to make the coordinated functional expression more complex Enzyme systems, gene clusters from metagenome and cDNA banks as well as whole ones Biosynthetic pathways in various Gram-negative bacteria allows.

• – Bereitstellen von mindestens zwei Gen-Kassetten, die Sequenzen aufweisen, welche die Übertragung und Integration eines flankierten Nukleinsäuremoleküls sowie die Expression aller Gene des flankierten Nukleinsäuremoleküls vermitteln;
• – Erzeugung eines rekombinanten Transposons durch Verknüpfung der Gen-Kassetten mit einem Nukleinsäuremolekül, das mindestens ein Gencluster umfasst, wobei mindestens eine erste Gen-Kassette stromaufwärts des Genclusters und mindestens eine zweite Gen-Kassette stromabwärts des Genclusters angehängt wird;
• – Einbringen des Transposons in eine Rezipientenzelle, wobei sich das Transposon selbstständig in das Genom der Rezipientenzelle integriert; und
• – Koordinierte Expression aller Gene des Genclusters in der Rezipientenzelle.

The object is achieved according to the invention by a method of the type mentioned at the outset, which comprises the following steps:

• Providing at least two gene cassettes having sequences which mediate the transfer and integration of a flanked nucleic acid molecule as well as the expression of all genes of the flanked nucleic acid molecule;
• Generating a recombinant transposon by linking the gene cassettes to a nucleic acid molecule comprising at least one gene cluster, wherein at least one first gene cassette is added upstream of the gene cluster and at least one second gene cassette downstream of the gene cluster;
• - introducing the transposon into a recipient cell, whereby the transposon integrates independently into the genome of the recipient cell; and
• Coordinated expression of all genes of the gene cluster in the recipient cell.

To overcome the mentioned problems in the known methods, an advantageous method for the cloning, integration and expression of large gene clusters in biotechnologically manageable expression hosts is provided according to the invention, which (i) allows the restriction endonuclease-independent one-step cloning of large gene clusters, (ii) the stable establishment allows the recombinant expression cassette in any Gram-negative bacteria, and (iii) allows complete expression of all target genes independent of the original promoters and transcription terminators. The targeted or randomized expression of host-foreign gene clusters in bacterial host strains with broad metabolic activities (the so-called "pathway engineering") allows the synthesis of new, bio technologically relevant substances. As a result, new bacterial expression and production strains can be produced for use in white biotechnology.

In a particularly advantageous embodiment of the method according to the invention, it is provided that the expression of all genes of the gene cluster in the recipient cell takes place with the aid of the T ₇ RNA polymerase.

The Linking the gene cassettes can be done, for example, by homologous recombination occurs. Alternatively, though also other genetic engineering in vitro methods of integration of the gene cassettes are applied to the target nucleic acid.

The Introduction of the recombinant transposon into the recipient cells can be done for example by conjugation. It can but alternatively also other transformation methods used become.

• – mindestens eine Promotor-Sequenz; und
• – mindestens ein Transpositionselement.

The stated object is further achieved by providing at least one gene cassette for cloning at least one gene cluster, wherein the gene cassette comprises the following elements:

• At least one promoter sequence; and
• - At least one transposition element.

The Gene cassette according to the invention is an integral part a universal expression system that uses combined expression many target genes in different bacterial expression hosts allowed and in particular for the implementation the method of the invention is suitable. The gene cassette allows targeted induction and controlled overexpression clustered target genes and allows the uncomplicated and Time-efficient cloning of large DNA fragments by sequence-specific Recombination processes. Furthermore, the inventive allows Gene cassette rapid production of suitable bacterial expression strains through the targeted transmission of a recombinant Transposons and its independent integration into the Host chromosome.

In a particularly advantageous embodiment of the gene cassette, the promoter sequence comprises the recognition and binding site for the T ₇ RNA polymerase. Furthermore, the gene cassette may additionally comprise at least one transposase gene, preferably the tnp gene. In a particularly advantageous embodiment of the invention, the transposition element may be an OE-L or an OE-R element. The gene cassette according to the invention may advantageously also additionally comprise at least one reporter gene and / or at least one selection marker.

In a particularly advantageous embodiment of the invention, the gene cassette comprises an antibiotic resistance gene with its own promoter, a reporter gene without its own promoter, a transposition element and a promoter for a non-bacterial RNA polymerase, preferably the T ₇ RNA polymerase.

alternative the gene cassette can be an antibiotic resistance gene with its own promoter, a reporter gene without its own promoter, one for a transposase coding gene, a transposition element and a promoter for include a non-bacterial RNA polymerase.

Within the gene cassette according to the invention, the antibiotic resistance gene may be, for example, a gene for tetracycline resistance (Tc ^R ) or for gentamicin resistance (Gm ^R ). The reporter gene may advantageously be a gene for kanamycin resistance (Km ^R ) or a gene coding for a fluorescent protein.

additionally the gene cassette of the invention may also be at least an origin of replication (oriT) and / or at least one operator sequence, preferably the lac operator.

Especially advantageous and preferred according to the invention a composition for cloning, integration and expression at least one gene cluster comprising at least two inventive Gene cassettes of the type described above comprises. The gene cassettes are constructed in such a way that they function mutually in their function complement and co-ordinate a coordinated and complete Enable expression of the gene cluster. The gene cassettes may be on both sides of the gene cluster to be expressed be attached so that they flank this and overall form a recombinant transposon into a suitable recipient cell can be introduced.

If two gene cassettes are contained in the composition according to the invention, these each comprise at least one promoter sequence and in each case at least one transposition element. Especially In an advantageous embodiment of the invention, each promoter sequence comprises the recognition and binding site for the T ₇ RNA polymerase. One of the two gene cassettes additionally comprises at least one transposase gene, preferably the tnp gene. In the case of the transposition elements, in a particularly advantageous embodiment of the invention it may be OE-L and / or an OE-R element. Each gene cassette advantageously additionally comprises at least one reporter gene and / or at least one selection marker. In a particularly advantageous embodiment of the invention, both gene cassettes each comprise an antibiotic resistance gene with its own promoter, a reporter gene without its own promoter, a transposition element and a promoter for a non-bacterial RNA polymerase, preferably the T ₇ RNA polymerase. In this case, one of the two gene cassette additionally comprises a gene coding for a transposase. Within the composition according to the invention with two gene cassettes, the respective antibiotic resistance gene may be, for example, a gene for tetracycline resistance (Tc ^R ) or for gentamycin resistance (Gm ^R ). The respective reporter gene may advantageously be a gene for kanamycin resistance (Km ^R ) or a gene coding for a fluorescent protein. In addition, one of the two gene cassettes may also comprise at least one origin of replication (oriT) and / or at least one operator sequence, preferably the lac operator. In this way, a composition is formed in which the two gene cassettes complement each other in an optimal manner and which enables efficient implementation of the method according to the invention.

According to the invention furthermore, a vector for cloning, integration and expression at least provided a gene cluster, wherein the vector at least one comprises gene cassette according to the invention. The vector may advantageously but also two or more gene cassettes include.

• a) Nukleotidsequenz, welche die Gen-Kassette nach einem der Ansprüche 5 bis 16 umfasst;
• b) Nukleotidsequenz, welche zwei Gen-Kassetten nach einem der Ansprüche 5 bis 16 umfasst;
• c) Nukleotidsequenz, welche den Vektor nach Anspruch 18 oder 19 umfasst;
• d) Nukleotidsequenz, welche die Sequenz gemäß SEQ ID NR. 13 und/oder 14 umfasst;
• e) Nukleotidsequenz, welche sich von der Nukleotidsequenz gemäß a), b), c) oder d) durch den Austausch zumindest eines Codons gegen ein synonymes Codon unterscheidet;
• f) Nukleotidsequenz, deren komplementärer Strang mit den Nukleotid-sequenzen gemäß a), b), c) oder d) hybridisiert;
• g) Nukleotidsequenz, welche zu mindestens 85%, vorzugsweise 90%, insbesondere 95%, mit der Nukleotidsequenz gemäß a), b), c) oder d) identisch ist;
• h) Nukleotidsequenz, welche dem komplementären Strang der Nukleotid-sequenzen gemäß a) bis g) entspricht.

The stated object is also achieved by the provision of a recombinant nucleic acid molecule which comprises at least one nucleotide sequence for the synthesis of at least one polypeptide, wherein the nucleotide sequence is selected from the group consisting of:

• a) Nucleotide sequence comprising the gene cassette according to any one of claims 5 to 16;
• b) A nucleotide sequence comprising two gene cassettes according to any one of claims 5 to 16;
• c) a nucleotide sequence comprising the vector of claim 18 or 19;
• d) nucleotide sequence which contains the sequence according to SEQ ID NO. 13 and / or 14;
• e) nucleotide sequence which differs from the nucleotide sequence according to a), b), c) or d) by the exchange of at least one codon for a synonymous codon;
• f) nucleotide sequence whose complementary strand hybridizes with the nucleotide sequences according to a), b), c) or d);
• g) nucleotide sequence which is identical to at least 85%, preferably 90%, in particular 95%, with the nucleotide sequence according to a), b), c) or d);
• h) nucleotide sequence which corresponds to the complementary strand of the nucleotide sequences according to a) to g).

According to the invention a kit for cloning, integration and expression at least a gene cluster provided which at least one inventive Includes gene cassette. Preferably, the kit comprises two or more gene cassettes.

The The invention also encompasses cells which contain at least one invention Gene cassette, the vector according to the invention and / or the nucleic acid molecule of the invention contained as well as cell cultures, which includes these cells.

The Invention will be described below with reference to examples and illustrations exemplified in more detail.

Brief description of the figures

1 : Schematic layout of the L and R IVAC cassettes. Both cassettes each have a selection marker with its own promoter (L-IVAC: the tetracycline resistance, Tc ^R , R-IVAC: the gentamicin resistance, Gm ^R ) and a promoterless reporter gene (L-IVAC: the kanamycin resistance, Km ^R ; R-IVAC: the yellow fluorescent protein, YFP). In addition, both IVAC cassettes each contain a T ₇ promoter and transposition elements (the DNA elements OE-L and OE-R and the transposase-encoding gene tnp (Tnp)). The T ₇ promoter of the R-IVAC cassette is under the control of a lac operator. The L-IVAC cassette also has an origin of transfer (oriT). The IVAC cassettes are flanked by the recognition sequences of the restriction endonucleases Scal and Smal, respectively. Recognition sequences of the homing endonucleases I-SceI and I-CeuI are also integrated in the IVAC cassettes.

2 : Schematic representation of the IVAC system. In the first step, the two IVAC cassettes (white rectangles) are integrated next to a gene cluster (gray arrows) by homologous recombination. The homologous regions are symbolized by the striped squares (step 1). In the second step, the labeled gene cluster is transferred by conjugation into a recipient. In the recipient cell, the gene cluster is transferred together with the IVAC cassettes (white squares) from the source vector (eg a BAC) into the recipient's chromosomal DNA (step 3) and expressed (step 4).

3 : Construction of the L-IVAC cassette. (A) Schematic structure of the L-IVAC cassette. (B) Schematic representation of the construction of the L-IVAC cassette. By PCR, the genes coding for antibiotic resistance and oriT were amplified and subsequently cloned into the vectors pUC18 and pUCPSK, respectively (step 1). The oligonucleotide containing the insertion sequence OE-L and the cleavage site for the enzyme I-CeuI was cloned into the recombinant plasmid pL-IVAC1 (step 2). The resulting vector was designated pL-IVAC2. The L-IVAC cassette was completed by cloning the oriT and the tetR gene into the vector pL-IVAC2 (step 3).

4 Construction of the R-IVAC cassette. (A) Schematic structure of the R-IVAC cassette. (B) Schematic of the construction of the R-IVAC cassette. The PCR products were cloned into the vectors pUC19 or pSVB10 and then analyzed by functional tests and sequencing. By hydrolysis of the vectors with BamHI and BclI or BglII, two components of the R-IVAC cassette (the gene YFP and the gentamycin resistance gene) were re-isolated from their vectors and successively inserted into the vector pR-IVAC1.

5 : RT-PCR for the detection of the gfp transcript in E. coli BL21 (DE3) with the plasmids R-IVAC> GFP or L-IVAC> GFP. The expression of the T ₇ polymerase was induced in E. coli with 1 mM IPTG. For the detection of the T ₇ polymerase-dependent transcription of gfp, E. coli was grown as a control with the corresponding plasmids even without the addition of IPTG. As a positive control and internal standard, the gentamicin resistance gene (Gm ^R ) was used.

6 : IVAC-dependent expression of the fluorescent marker GFP in E. coli BL21 (DE3). (A) L-IVAC-mediated GFP expression. (B) L-IVAC-mediated GFP expression. In the respective E. coli strains, the expression of the GFP was specifically induced by addition of 1 mM IPTG.

7 : RT-PCR for detecting the transcription of the lac operon in R. capsulatus with the transposon R> lacZYA. The transcripts of the genes lacZ, lacY and lacA could be detected in R. capsulatus after induction of transfection with fructose by RT-PCR.

8th : RT-PCR for detecting the transcription of the lac operon in P. putida with the transposon R> lacZYA. The transcripts of the genes lacZ, lacY and lacA could be detected in P. putida after induction of the transcription with IPTG by RT-PCR.

9 : RT-PCR for the detection of antisense mRNA synthesis in R. capsulatus with the transposon R> lacZYA. The complementary transcripts of the genes lacZ, lacY and lacA could be detected in R. capsulatus after induction of transfiption with fructose by RT-PCR.

10 : RT-PCR for the detection of antisense mRNA synthesis in P. putida with the transposon R> lacZYA. The complementary transcripts of the genes lacZ, lacY and lacA could be detected in P. putida after induction of the transcription with IPTG by RT-PCR.

11 : Detection of β-galactosidase activity in R. capsulatus. The activities of β-galactosidase are given in Miller units. It is shown the activity in R. capsulatus wild type (WT) and in the heterologous host strains without T7 polymerase (-pML5-P _fru -T7) or with T7 polymerase (+ pML5-P _fru -T7).

12 : Detection of β-galactosidase activity in P. putida. The activities of β-galactosidase are given in Miller units. It is the activity in P. putida wild type (WT) shown as well as in the heterologous host strains without T7 polymerase (-pML5-T7) or with T7 polymerase (+ pML5-T7).

13 : Plasmid map of the plasmid pIC20HRL.

14 : Plasmid map of the plasmid pR-IVAC> GFP.

15 : Plasmid map of the plasmid pL-IVAC> GFP.

16 : Plasmid map of the plasmid pL> lacZYA.

17 : Plasmid map of the plasmid pR> lacZYA.

Different and preferred Embodiments of the invention

• 1) Das IVAC-System ist ein universelles Expressionssystem, das die kombinierte Expression vieler Zielgene in unterschiedlichen bakteriellen Expressionswirten erlaubt.
• 2) Es erlaubt die gezielte Induktion und kontrollierte Überexpression geclusterter Zielgene.
• 3) Es ermöglicht die unkomplizierte und zeiteffiziente Klonierung großer DNA-Fragmente durch sequenzspezifische Rekombinationsprozesse.
• 4) Die Verwendung des IVAC-Systems erlaubt eine schnelle Erzeugung geeigneter bakterieller Expressionsstämme durch die zielgerichtete Übertragung des rekombinanten Transposons und dessen eigenständige Integration in das Wirtschromosom.

To carry out the process according to the invention, a novel composition, the so-called "in vivo auto cloning and expression" system (IVAC), was created for the universal heterologous expression of clustered genes. [IV Das] The IVAC system has the following properties:

• 1) The IVAC system is a universal expression system that allows the combined expression of many target genes in different bacterial expression hosts.
• 2) It allows the targeted induction and controlled overexpression of clustered target genes.
• 3) It allows the uncomplicated and time-efficient cloning of large DNA fragments by sequence-specific recombination processes.
• 4) The use of the IVAC system allows rapid production of suitable bacterial expression strains by the targeted transfer of the recombinant transposon and its independent integration into the host chromosome.

Overview of the structure the composition according to the invention (IVAC system)

The IVAC system is based on two cassettes, the L-IVAC and the R-IVAC cassette. The structure of the cassettes is in 1 shown schematically. The IVAC cassettes are composed of various individual components. Both cassettes each contain a selection marker (the antibiotic resistance genes for Tc and Gm), a reporter gene (promoterless genes coding for the yellow fluorescent protein (YFP) and kanamycin resistance (Km ^R )), a T ₇ promoter and Transposition elements (OE-L, OE-R and a transposase gene). The L-IVAC cassette also has an origin of replication (oriT) and a lac operator in the R-IVAC cassette.

Overview of how it works the composition of the invention

The two IVAC cassettes are integrated by homologous recombination upstream and downstream of a target gene cluster, which preferably already resides on an appropriate vector (eg, a BAC) ( 2 , Step 1). With the help of the IVAC system, the following steps will enable the transfer of the gene cluster into a bacterial expression strain by conjugation. The specific arrangement of the two IVAC cassettes and of the gene cluster surprisingly produces a functional recombinant transposon which, after the transfer, is integrated independently into the chromosome of the recipient strain ( 2 , Steps 2 and 3). After integration, the genes of the target cluster can be transcribed by the T ₇ RNA polymerase ( 2 , Step 4). The advantage of T ₇ RNA polymerase compared to bacterial polymerases is that it does not recognize bacterial transcription terminators and thus the complete transcription of all genes of a cluster is possible independently of transcription terminators ( Macdonald et al., 1992 ). Unexpectedly, all genes of a gene cluster can be expressed from both sides and thus independently of their orientation via the oppositely arranged T ₇ promoters of the IVAC cassettes ( 2 , Step 4).

Construction of IVAC cassettes

• – Sie durften keine Erkennungssequenzen für Restriktionsendonukleasen aufweisen, die bei späteren Klonierungsschritten stören könnten.
• – Sie mussten von den kompatiblen Erkennungssequenzen der Restriktionsendonukleasen BamHI, BclI oder BglII flankiert werden.

The L-IVAC cassette consists of a total of six, the R-IVAC cassette of eight functional genetic elements. Due to the complex structure of the IVAC cassettes, a special cloning strategy was needed to construct the cassettes. The DNA fragments comprising the individual elements were partially amplified by PCR, in part being components of synthesized oligonucleotides. For the cloning of the individual components of the IVAC cassettes they had to possess the following properties:

• - They should not have recognition sequences for restriction endonucleases, which could interfere with later cloning steps.
• They had to be flanked by the compatible recognition sequences of the restriction endonucleases BamHI, BclI or BglII.

By combining these compatible interfaces, the individual compo IVAC cassettes and at the same time delete the recognition sequences between them. The underlying cloning strategies are described in the 3 (Construction of the L-IVAC cassette) and 4 (construction of the R-IVAC).

Detection of the activity of the integrated in the IVAC tapes T ₇ promoters

• (1) Die Anwesenheit des T₇-Promotors führte unabhängig von der verwendeten IVAC-Kassette nach Zugabe von IPTG in den E. coli Expressionsstämmen zu einer signifikanten Transkription des GFP-Gens. In beiden Fällen (5: R-IVAC>GFP +IPTG und L-IVAC>GFP +IPTG) konnte bereits nach acht Zyklen das entsprechende Transkript nachgewiesen werden.
• (2) Unter uninduzierten Bedingungen (5: R-IVAC>GFP –IPTG und L-IVAC>GFP –IPTG) war in den entsprechenden Expressionsstämmen eine deutlich verminderte Transkription des Reportergens zu verzeichnen. Im Vergleich zur induzierten Expression liegt in diesem Fall nur 2–3% des gfp Transkripts vor.
• (3) Um ausschließen zu können, dass die Synthese der detektierten GFP PCR-Produkte auf eine Verunreinigungen der isolierten mRNA mit Plasmid-DNA zurückzuführen ist, wurde die aus den E. coli-Stämmen isolierte RNA, die nicht mittels reverser Transkriptase in ihre cDNA umgeschrieben wurde, direkt als Template in der RT-PCR eingesetzt. Sogar nach 30 PCR-Zyklen konnte in diesen Kontrollproben kein PCR-Produkt nachgewiesen werden (Daten nicht gezeigt). Somit konnte eine Kontamination der isolierten RNA durch Plasmid-DNA ausgeschlossen werden.

To check whether the localized in the IVAC tapes T ₇ promoters are functional, the individual IVAC cassettes were cloned upstream of a promoterless gfp gene. The resulting GFP expression plasmids are referred to below as pL-IVAC> GFP and pR-IVAC> GFP ( 14 and 15 ). To check whether the GFP gene can be expressed starting from the IVAC-T ₇ promoters, the E. coli T ₇ expression strain BL21 (DE3) was transformed with the generated plasmids and the expression of the reporter protein by means of RT-PCR ( 5 ) and GFP fluorescence measurements ( 6 ). For this purpose, the generated expression strains were grown to the early logarithmic growth phase, and the T ₇ polymerase-dependent expression of the GFP reporter gene with IPTG induced (+ IPTG). The controls were identical cultures in which GFP expression was not induced (-IPTG). The results of the RT-PCR are in the 5 and can be summarized as follows:

• (1) The presence of the T ₇ promoter, regardless of the IVAC cassette used, resulted in significant transcription of the GFP gene after addition of IPTG in the E. coli expression strains. In both cases ( 5 : R-IVAC> GFP + IPTG and L-IVAC> GFP + IPTG), the corresponding transcript was detected after only eight cycles.
• (2) Under uninduced conditions ( 5 : R-IVAC> GFP-IPTG and L-IVAC> GFP-IPTG) significantly reduced transcription of the reporter gene was found in the corresponding expression strains. Compared to induced expression, only 2-3% of the gfp transcript is present in this case.
• (3) In order to rule out that the synthesis of the detected GFP PCR products is due to contamination of the isolated mRNA with plasmid DNA, the RNA isolated from the E. coli strains, which did not convert into its cDNA by reverse transcriptase was rewritten, used directly as a template in the RT-PCR. Even after 30 cycles of PCR, no PCR product could be detected in these control samples (data not shown). Thus, contamination of the isolated RNA by plasmid DNA could be excluded.

In order to be able to show that GFP can be functionally expressed in E. coli using the IVAC expression cassettes, GFP-specific fluorescence was then quantified in protein extracts of the corresponding expression strains. The results of this fluorescence measurement are in 6 shown. In the E. coli expression strains carrying the plasmid pL-IVAC> GFP ( 6A ) or pR-IVAC> GFP ( 6B ), only after induction of GFP expression (+ IPTG) could fluorescence be detected at a specific wavelength of 410 nm. In the absence of the inducer (-IPTG), no GFP fluorescence was detectable. From this example it could thus be shown that the T ₇ promoters of the two IVAC cassettes are functional and a T ₇ polymerase-dependent overexpression with the generated IVAC cassettes is possible.

The IVAC-mediated integration and heterologous Expression of a gene cluster in Pseudomonas putida and Rhodobacter capsulatus using the example of the E. coli lac operon

Gfp based transcriptional fusions with the gene could be shown that with the IVAC cassettes a _T7 polymerase-dependent expression of genes can be achieved. In the following, it was examined whether it is also possible with the aid of the IVAC cassettes to transfer a suicide plasmid with a gene cluster to be expressed by conjugation into a heterologous host strain, to transfer the cluster by transposition into the genome of the host and subsequently with the aid of to express the T ₇ polymerase.

To test these properties of the IVAC system, the promoterless lac operon from E. coli should be heterologously expressed in Rhodobacter capsulatus and Pseudomonas putida. For this purpose, two plasmids were constructed, which are referred to below as pL> lacZYA and pR> lacZYA ( 16 and 17 ). Both plasmids contain both the lac operon from E. coli and both IVAC cassettes. They differ only in the arrangement of the IVAC cassettes. In the plasmid pL> lacZYA, the IVAC cassettes are arranged such that the T ₇ polymerase transcribes the lac operon from the T ₇ promoter of the L-IVAC cassette, in the plasmid pR> lacZYA transcribing the T ₇ polymerase starting from Promoter of the R-IVAC cassette the operon. Since R. capsulatus and P. putida do not possess a T ₇ polymerase gene, this is established in the strains after IVAC transposition via derivatives of the replicative broad host range plasmid pML5. The plasmid pMI5T7 carries the gene for the T ₇ polymerase under the control of the lac promoter and was used in P. putida. In the plasmid pML5-P _fru -T7 the polymerase gene is under the control of the Rhodobacter specific Fructose promoter.

Transmission of the lac operon in the heterologous host strains

Around to check if the transmission and Establishment of a gene cluster by means of conjugation and transposition using the new IVAC system the plasmids pL> lacZYA and pR> lacZYA first in the conjugation-optimized donor strain E. coli S17-1 transformed. The recipients were Gram-negative bacteria R. capsulatus and P. putida. Conjugation and transposition were as described in the appendix. Because the to be transferred Plasmids possess a gentamicin resistance gene, the recipient strains, which is obtained by conjugation of a plasmid and by transposition integrated into their genome from the respective wild-type strain be distinguished by selection for the antibiotic.

The experiments showed that the conjugation and transposition of the plasmids using the IVAC cassettes could be mediated in both heterologous host strains. Surprisingly, the transposition rate of the recombinant transposons (L> lacZYA and R> lacZYA in R. capsulatus: 2 × 10 ^-6 and in P. putida: 7 × 10 ^-5 ) did not differ significantly from the transposition rate of the natural Tn5 transposon (transposition rate : about 6.5 × 10 ^-5 (Naumann & Reznikoff, 2002)). The comparison with the natural transposon Tn5 thus shows that the recombinant IVAC transposons could be easily transferred to the selected host strains and integrated into their chromosomes.

Detection of T ₇ polymerase-dependent transcription of the lac operon in the heterologous host strains.

After the successful integration of the lac operon and the IVAC cassettes in the genome of the heterologous hosts R. capsulatus and P. putida T was checked ₇ polymerase dependent transcription of the lac operon by RT-PCR. In the heterologous host strains, the expression of the T ₇ polymerase and thus of the lac genes was induced by the addition of fructose (R. capsulatus) or IPTG (P. putida). Subsequently, the mRNA of these strains was isolated. In the subsequent reverse transcription, primers were used to detect the transcripts of lacZ, lacY and lacA and the resulting cDNA was used as template in an RT-PCR. The results of the RT-PCR are in the 7 (Capsulatus R.) and 8 (P. putida). As a transcript control, the isolated mRNA from R. capsulatus or P. putida was also used directly (ie without previous cDNA synthesis) as a template for the RT-PCR also in this case.

As in 7 can be clearly seen, a complete transcription of the lac operon was detected in R. capsulatus with the transposon R> lacZYA by RT-PCR. In this case, a specific PCR product is detectable after cycle 15 (lacZ), 17 (lacA) and 18 (lacY). There is thus transcript from all genes of the operon. Even with the L> lacZYA transposon, all lac transcripts can be detected in comparable amounts (not shown). The negative control, the strain with integrated transposon but without the T ₇ -Polymerase carrying plasmid pMl5-P _Fru T7, has no transcript (not shown).

8th shows the results of RT-PCR with R> lacZYA in P. putida. The signals of the lacZ, lacY and lacA transcripts are clearly detectable in the 15th, 17th and 18th cycle. As already shown for R. capsulatus, complete expression of the transferred lac genes was also achievable in P. putida with the corresponding L> lacZYA construct (not shown). The P. putida strains without the plasmid pMI5T7 but with integrated transposon in contrast to R. capsultus transcript of the genes lacZ, Y, A in low concentration (not shown). By adding the T ₇ polymerase, however, the transcript amount of the genes is significantly increased. In P. putida, Pseudomonas promoters located upstream of the IVAC-lacZYA transposon on the host chromosome can induce low expression of the test genes.

Detection of antisense transcripts in R. capsulatus and P. putida

Using the example of the lac operon of E. coli could be detected so far that a "labeled" by IVAC cassettes gene cluster by conjugation and transposition can be successfully established in bacterial host strains. After the induction of T ₇ polymerase expression could also In both tested expression hosts, the lac genes of the test gene cluster are transcribed, however, since the genes of complex gene clusters, unlike the lac operon, are often not in an orientation, the transcription of the genes flanked by the two IVAC cassettes must also be in principle In the following, RT-PCR should be used to investigate to what extent antisense transcripts of the lac gene cluster in the expression strains generated by the IVAC technology.

In the 9 and 10 the results of these investigations are shown. Astonishingly, both in R. capsulatus ( 9 ) as well as in P. putida ( 10 ) T ₇ expression strain after the establishment of the R> lacZYA transposon significant amounts of the respective antisense transcripts are detected. Also in this case, the synthesis of RNA is independent of the arrangement of IVAC cassettes (not shown). To check whether the synthesis of the antisense RNA is dependent on the T ₇ polymerase, the bacterial strains with integrated transposon but without T ₇ polymerase were used in the RT-PCR. They have no transcript (R. capsulatus) or low-concentration transcript levels (P. putida) (not shown). In order to be able to demonstrate that even in these experiments only the cDNA produced by the transcripts and not the chromosomal DNA delivered the signals in the RT-PCR, appropriate controls (RNA samples without reverse transcriptase) were used. As expected, no amplicon could be generated in these cases.

through The IVAC expression system will include both sense and antisense RNA of the gene cluster to be expressed in the tested bacterial Host trunks formed. The genes of a gene cluster can thus amazingly, by means of the IVAC system independently from their orientation and without mutual obstruction of the transcribed opposite polymerases are transcribed.

Detection of β-galactosidase activity in the heterologous host strains R. capsulatus and P. putida

After successfully detecting the sense transcripts and antisense transcripts of the lac genes in the heterologous hosts R. capsulatus and P. putida, the question arose as to whether a possible hybridization of the mutually complementary mRNA molecules can inhibit translation. In order to detect the synthesis of β-galactosidase in all expression strains used, an enzyme activity test was carried out with the substrate O-nitrophenyl-β-D-galactopyranoside (ONPG) for this purpose. 11 shows the activity of the expressed β-galactosidase in R. capsulatus (L> lacZYA, pML5 _fru-P T7), R. capsulatus (R> lacZYA, pML5 _fru-P T7). In both cases, unexpectedly, a strong β-galactosidase activity of max. 700 miller units are detected. In the P. putida expression strains even enzyme activities of max. Reached 900 miller units ( 12 ). Since neither the R. capsulatus nor the P. putida wild-type strain can synthesize an active β-galactosidase, no corresponding enzyme activity could be detected as expected in the wild-type controls (WT) ( 11 and 12 ). In addition, by determining the enzyme activities in the L> lacZYA and R> lacZYA strains lacking the T ₇ polymerase-encoding plasmid, it was possible to examine whether Rhodobacter or Pseudomonas promoters upstream of the IVAC-lacZYA transposon on the host chromosome which can bring about expression of the test genes. However, such promoter activity was not observed in R. capsultus and only to a minor extent in the P. putida expression strain.

There clearly in the IVAC-bearing expression strains a β-galactosidase activity could be proved, surprisingly excluded be that synthesis of RNA in sense and antisense direction leads to an inhibition of translation.

The method according to the invention and the composition according to the invention consequently enable easy labeling of a heterologous gene cluster. Through the one-step transfer and integration of the recombinant IVAC transposon into the chromosome of a bacterial host strain, an expression strain is generated which allows the targeted expression of all genes of the transferred cluster. Appendix / Media Cultivation of E. coli and P. putida media LB liquid medium ( Sambrook et al., 1989 ): tryptone 10g NaCl 10 g yeast extract 5 g A. least. ad 1000 ml LB agar: agar 15 g LB medium ad 1000 ml

The growth of the bacteria was, unless otherwise stated, at 37 ° C (E. coli) and 30 ° C (P. putida). Overnight cultures (ÜK) were incubated for at least 16 h. Cultures up to 5 ml in volume were incubated in the test tube on a breeding roller, larger cultures in the Erlenmeyer flask (culture volume: maximum 1 / 5-1 / 10 of the vial volume) on a rotary shaker at 200 rpm. Precultures were grown in LB medium and inoculated with either a single colony or 1/100 volume from a freezing culture. Main cultures were inoculated to a cell density corresponding to an OD _580nm of 0.05. The cell densities of cultures were determined by turbidity measurements in a spectrophotometer at a wavelength of 580 nm against the corresponding medium as a reference. An OD _580nm of 1 corresponds to a cell number of about 2 x 10 ⁹ per ml of culture. For freezing cultures, 1.8 ml of a ÜK were mixed with 138 μl of DMSO and stored at -80 ° C. Cultivation of R. capsulatus media PY-rich medium A dist ad 1000 ml Beato Peptone ad 10.0 g yeast extract 0.5 g agar 15 g 1M MgCl2 2 ml PY medium ad 1000 ml 1M MgCl2 2 ml 0.5% FeSO4 2.4 ml PY agar agar 15 g PY medium ad 1000 ml RCV minimal medium: 10% DL malate 40.0 ml 20% MgSO ₄ 1.0 ml 7.5% CaCl ₂ 1.0 ml 1% EDTA 2.0 ml 0.5% FeSO ₄ 2.4 ml 0.1% thiamine 1.0 ml Trace element solution 1.0 ml 1 M phosphate buffer 9.6 ml A. least ad 1000 ml pH 6.8 RCV + N: 10% (NH ₄ ) ₂ SO ₄ 10.0 ml RCV ad 1000 ml Trace element solution for RCV: MnSO ₄ (X1h ₂ O) 0.4 g H ₃ BO ₃ 0.7 g Cu (NO ₃ ) ₂ (x3H ₂ O) 0.01 g ZnSO ₄ (X7H ₂ O) 0.06 g NaMoO ₄ (x ₂ H ₂ O) 0.02 g A. least ad 1000 ml

Phototrophic cultivation of R. capsulatus under high light conditions

R. capsulatus was preferably anaerobically grown in the light (photoheterotrophic). Anaerobic cultivation was carried out as a single colony smear on solid medium or in liquid medium in EPGs (up to 1.5 ml) in a special anaerobic incubation pot using the Gas-Pack Anaerobic System (BBL). Larger volumes of culture were grown in gas-tight sealable tubes (Hungates) or Schott bottles fumigated with argon or N ₂ using a septum in the lid. Under these growth conditions, R. capsulatus was incubated for three days in the high light (six bulbs a 60 W, equivalent to 2500 lux).

Aerobic seedling of R. capsulus in the dark

The Incubation of R. capsulatus single colony smears was also possible under aerobic conditions in the incubator (in darkness).

With Cell material from anaerobically grown plate cultures could R. capsulatus be aerobically cultured in liquid medium. This was done incubation in a test tube in the dark on a dark Brood Roller.

Standard mini-preparation

The isolation of plasmid DNA from E. coli and P. aeruginosa was carried out according to the method based on the principle of alkaline lysis developed by Birnboim & Doly (1979). Mix I (pH 8.0): 50 mM glucose 50 mM Tris-HCl 10mM EDTA Mix II: 200mM NaOH 1% (w / v) SDS Mix III (pH 4.8): 3 m potassium acetate 1.8 m formate TE buffer (pH 8.0): 10mM Tris-HCl 1 mm EDTA

bacterial cells from 2 ml of a test sample grown under selective pressure (2.6.3) were harvested by centrifugation (3 min, 13000 rpm, EA, RT). After complete removal of the culture supernatant the sediments were resuspended in 300 μl Mix I and incubated for 1 min at RT. Subsequently were Add 300 μl of Mix II and mix with the samples. After addition of 300 μl Mix III and 10 min incubation on Ice were the resulting protein-SDS precipitates, the precipitated chromosomal DNA as well as cell debris Centrifugation (15 min, 13000 rpm, EA, RT) sedimented. The clear, supernatant containing the plasmid DNA was removed with 10 ul of ribonuclease A (2 mg / ml) and 30 min incubated at 37 ° C. After subsequent ethanol precipitation or isopropanol precipitation, the plasmid DNA was in 30-50 μl 0.1 × TE buffer and until further use stored at -20 ° C.

Hydrolytic cleavage of DNA with restriction endonucleases

The hydrolytic cleavage of DNA with restriction endonucleases under the conditions recommended by the manufacturer in each case optimal reaction buffers. The type II restriction endonucleases used here bind to a short sequence of nucleotides and cleave DNA molecules within this recognition sequence. 5 μg per μg of DNA used to 10 U of the corresponding enzyme and the restriction mixture Incubated for 1-2 h at 37 ° C. The restriction endonucleases were inactivated prior to ligations by heat treatment, for that the mixtures were incubated at 65 ° C for 20 min.

Ligation of DNA fragments

For ligation of DNA fragments, T4 ligase was used in the reaction buffer provided by the manufacturer. T4 ligase catalyzes the ATP-dependent formation of phosphodiester bonds between adjacent 3'-hydroxy and 5'-phosphate ends both between two compatible, sticky end and blunt end. In a reaction volume of 10-20 μl, the fragment DNA was mixed with the vector DNA in a molar ratio of at least 3: 1 and with 1 U T4 polymerase and incubated either for 2 h at RT or üN at 11 ° C. In the blunt end ligation, the reaction batches were additionally supplemented with PEG 4000 (Fermentas) at a final concentration of 5%. For subsequent transformations, the ligation mixtures were diluted 1: 4 with TMF buffer. Transformation of bacteria with plasmid DNA Preparation of transformation competent cells Transformation buffers: 100 mM CaCl ₂ 50 mM RbCl ₂ 40 mM MnCl ₂ Mg ²⁺ mix: 1 m MgCl ₂ 1 m _MgSO4

In a sterile 100 ml Erlenmeyer flask, 20 ml LB medium was mixed with 0.4 ml Mg ²⁺ mix and inoculated with the amount of an overnight E. coli _culture so that the cell density corresponded to an OD _{580 nm} of 0.05. This culture was incubated on a shaker (200 rpm) at 37 ° C until it was in the logarithmic growth phase (DD _580nm = 0.5-0.8). Subsequently, the culture was aliquoted in 2 ml batches, the cells were sedimented by centrifugation (2 min, 8000 rpm, EA, 4 ° C). The pellet was resuspended in 1 ml of ice-cold transformation buffer (TMF buffer) and the mixtures incubated on ice for 1 h. After subsequent centrifugation (2 min, 8000 rpm, EA, 4 ° C), the pellets were each taken up in 200 μl of ice-cold TMF buffer. Until further use, the batches were stored on ice or after addition of glycerol at a final concentration of 20% (v / v) at -80 ° C.

Transformation of E. coli

to Transformation were 200 ul transformation competent Cells with 20 μl of a ligation batch or about 100 ng prepared plasmid DNA mixed, at least 30 min on Incubated ice and then for 90 s one Heat shock exposed at 42 ° C. After addition of 700 .mu.l LB medium was followed by phenic expression, if not stated otherwise, at 37 ° C on the breed roller, depending on Antibiotic resistance lasted 1-3 h. 100 μl of this Approach was plated on appropriate Selektivagar, the the remaining batch was centrifuged (2 min, 8000 rpm, EA, RT), the pellet was taken up in 100 μl of LB medium and also plated on selective agar. The negative control used was an approach transformation-competent cells, no plasmid DNA or no Ligation mixture was added.

Transfer of plasmid DNA by Conjugation and transposition

For the conjugative transfer of mobilizable plasmids, the method of "biparental mating" was used.The donor-applied E. coli strain S17-1 carries the tra genes of the RP4 plasmid ( Simon et al., 1986 ) stably integrated in the genome. The transposition of the plasmid into the genome of the recipient is catalyzed by the enzyme transposase.

Conjugation and transposition of plasmids from E. coli to P. putida

When Recipients served one of the P. putida strains KT2440. The Cultivation of P. putida and E. coli strains to be conjugated was carried out in LB medium under appropriate selection pressure üN at 30 ° C (P. putida) or 37 ° C (E. coli).

ever 0.5 ml of the recipient culture and the donor culture were in a EPG mixed and centrifuged. Subsequently, the Pellet washed with 1 ml of LB medium to remove the cells from the corresponding To get rid of antibiotics. The cells were centrifuged again (8000 rpm, EA, RT), resuspended in its residual supernatant and placed on a filter on LB medium. After an incubation period of 16 hours at 30 ° C, the filter was in an EPG with Given LB medium, cells were vortexed by the filter solved. It was a dilution series of this Cells are applied and plated on appropriate Selektivagar. to Contrast selection of the E. coli donor strain was Irgasan (Ciba Geigy, Basel) in a final concentration of 25 μg / ml.

Conjugation and transposition of plasmids from E. coli to R. capsulatus

The recipient was picked up from a fresh anaerobic stem plate in 5 ml minimal medium (RCV + N) and resuspended at 30 ° C. on the incubator. The plasmid-bearing E. coli S17-1 donor was selectively grown at 37 ° C. on an LB agar plate. From the donor plate single colonies of the logarithmic growth phase were harvested and resuspended in 1 ml of PY-Fe. 1 ml each of the recipient culture was mixed with 0.5 ml of the donor culture and centrifuged in an Eppendorf tube (5 min, 13,000 rpm, RT). The sediment was gently resuspended in the reflux. The cell suspension was then transferred to nitrocellulose filters and incubated at 30 ° C. on PY agar plates. The filters were resuspended in 1 ml minimal medium (RCV + N). From the bacterial culture, a dilution series was applied and different volumes were streaked onto selective medium. This was followed by a 3-day incubation in the incubator at 30 ° C.

Polymerase Chain Reaction (PCR)

• ( Mullis & Fallona, 1987 )

The polymerase chain reaction was used to amplify genes. By selecting corresponding oligonucleotides as starter molecules, restriction endonuclease recognition sequences were added upstream and downstream of the genes, which served for the targeted cloning of the amplified fragments into cloning vectors. The amplification was carried out using the thermostable DNA-dependent DNA polymerase from Pyrococcus furiosus. Tab. 2.5: Concentrations of the reaction components in PCR reactions with turbo-Pfu DNA polymerase and Pfu DNA polymerase reactant concentration Oligonucleotide I 5-10 pmol Oligonucleotide II 5-10 pmol Pfu polymerase buffer 1 × Pfu buffer dNTP mix 0.25 mM DNA polymerase 1-2.5 U DMSO 5mM Template DNA 50 ng

The Volume of a reaction mixture was 50 μl. The PCR reactions was in the PCR machine "Mastercycler Gradient" the Fa. Eppendorf performed.

fluorescence measurements

PBS buffer (pH 7.0)

• 4 mM KH ₂ PO ₄ ; 16 mM Na ₂ HPO ₄ ; 115 mM NaCl

Whole cell fluorescence measurements were performed in PBS buffer. For this purpose, aliquots corresponding to OD ₅₈₀ = 1 were taken from the respective expression cultures, pelleted and washed with PBS buffer. Subsequently, the cells were taken up in 1 ml PBS buffer. The cell suspensions thus obtained were used as stock solutions for the following fluorescence measurements. The fluorescence of the samples was measured with a fluorescence photometer ( Luminescence SSpectrometer LS 506, Perkin Elmer, Boston, USA ). Detection of β-galactosidase activity 4 × Z buffer 21.36 g Na ₂ HPO ₄ 11.04 g NaH ₂ PO ₂ 1.49 g KCL 0.49 g _MgSO4 ad 1 L Cell disruption buffer 1 ml 4 × Z buffer 14 μl β-mercaptoethanol 7.5 μl 20% SDS

ONPG solution

• 0.8 mg / ml ONPG; 14 μl / ml β-mercaptoethanol; in 1xZ buffer

Stop buffer

• 1M Na ₂ CO ₃

The Enzyme activity of β-galactosidase was measured with the Examined ONPG test. The ONPG test replaces the synthetic galactoside O-nitrophenol-β-D-galactopyranoside the natural Substrate of β-galactosidase. It gets through like lactose the galactoside permease is introduced into the cell and by the β-galactosidase hydrolyzed. This produces the two reaction products galactose and o-nitrophenol. The latter causes a yellowing, which can be measured photometrically. At first were determines the cell densities of each culture. 400 μl each the samples were mixed with 25 μl chloroform and 25 μl of the cell disruption buffer, resuspend and incubate for 5 min RT incubated. After the addition of 400 μl ONPG solution was added the time to yellowing of the samples was measured. By the addition of the stop buffer could terminate the reaction and the Yellowing of the samples can be measured photometrically.

mRNA quantification by means of real-time RT-PCR

To isolate total bacterial RNA, an aliquot of the bacterial culture in the exponential growth phase (OD ₅₈₀ = 0.9) was harvested (4000 rpm, 4 ° C, 5 min). The cell pellet was then either processed directly or stored at -80 ° C until further use ( Berstein et al., 2002 ; Khodursky et al., 2003 ). Freshly centrifuged or frozen pellets were processed with the RNeasy Mini Kit (Qiagen, Hilden). For this, the cells were resuspended in 350 μL of the RNeasy RLT buffer. After centrifugation (2 min, 13000 rpm), the supernatant (maximum 700 μl) was applied to the RNeasy columns to isolate the total RNA (Qiagen, Hilden). The silica gel-based membranes of the columns selectively bind single-stranded RNA molecules in the presence of absolute ethanol (250 μl) and a specific salt buffer concentration. This was followed by two more washes before the RNA was eluted from the column with two times 50 μl of RNase-free water. The prepared RNA was subjected to DNase digestion. For this purpose, the sample was incubated with 5 μl 10 × DNase buffer and 30 U RNase-free DNase (Promega) for 20 min at 37 ° C. Addition of the stop buffer was followed by inactivation of the enzyme for 10 minutes at 70 ° C. Stored RNA was stored at -20 ° C.

Synthesis of the cDNA

The Synthesis of the cDNA was carried out starting from 500 ng of total RNA, the by reverse transcription with reverse transcriptase (Omniscript Reverse transcriptase, Qiagen, Hilden) into cDNA. The RNA was pre-synthesized for 5 min at 68 ° C incubated and then cooled for 5 min on ice. Subsequently was the prepared master mix of reaction buffer, nucleotides and reverse transcriptase added. The synthesis was carried out for 1 h at 37 ° C. The synthesized cDNA was without further Purification used as a template in real-time PCR.

RealTime PCR

Real-time PCR was performed with the LightCycler realplex (Eppendorf). The reagents used were the components Quantitect SYBR Green I Kit (Qiagen, Hilden). The fluorescent dye SYBR Green I (Invitrogen, Karlsruhe) binds double-stranded DNA and has an absorption maximum at a wavelength of 521 nm. The table below lists the course of the RealTime PCR and the melting curve. 95 ° C 2 min denaturation 95 ° C 15 sec 55 ° C 30 sec 40 cycles 68 ° C 30 sec 95 ° C 15 sec melting curve 60-95 ° C 20 min

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It follows Sequence listing according to WIPO St. 25. This can of the official publication platform of the DPMA become.

QUOTES INCLUDE IN THE DESCRIPTION

This list The documents listed by the applicant have been automated generated and is solely for better information recorded by the reader. The list is not part of the German Patent or utility model application. The DPMA takes over no liability for any errors or omissions.

Cited non-patent literature

• - Wenzel & Müller, 2005 [0002]
• - Gustaffson et al., 2004 [0005]
• Lambalot et al., 1996 [0006]
• Drepper et al., 2005 [0007]
• - de Marco et al., 2007 [0007]
• - Wenzel et al., 2005 [0007]
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• - Kao et al., 1994 [0010]
• - Pfeifer et al., 2001 [0011]
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• - Lau et al., 2002 [0011]
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• Luminescence SSpectrometer LS 506, Perkin Elmer, Boston, USA [0091]
• Berstein et al., 2002 [0093]
• Khodursky et al., 2003 [0093]
• - Birnboim, HC. & Doly, J. (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA Nucleic. Acids Res. 7: 1513-1523 [0095]
• - Marco A., Deuerling E., Mogk A., Tomoyasu T., Bukau B. (2007) Chaperone-based procedure to increase the yields of soluble recombinant proteins produced in E. coli BMC Biotechnology 2007, 7; 32 [0095]
• - Drepper T., Arvani S., Rosenau F., Wilhelm S., Hunter KE. T7-RNA polymerase based system for the expression of functional hydrogenases in the phototrophic bacterium Rhodobacter capsulatus Biochem Society Transactions 33, part 1 [0095]
• Gustaffson, C., Govindarajan S., Minshull J. (2004) Codon bias and heterologous protein expression Trends Biotechnol 22: 346-353 [0095]
• - Julien B., Shah S. (2002) Heterologous expression of epothilone biosynthetic genes in Myxococcus xanthus Antimicrob Agents Chemother 46: 2772-2778 [0095]
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Claims (24)

Methods for cloning, integration and expression multiple genes of at least one gene cluster, wherein the method the following steps include: - Deploying at least two gene cassettes that have sequences that confer transmission and integration of a flanked nucleic acid molecule as well as the expression of all genes of the flanked nucleic acid molecule convey; - Generation of a recombinant transposon by linking the gene cassettes with a nucleic acid molecule, comprising at least one gene cluster, wherein at least a first Gene cassette upstream of the gene cluster and at least a second gene cassette attached downstream of the gene cluster becomes; Introduction of the transposon into a recipient cell, wherein the transposon is independent in the genome of Recipient cell integrated; and - Coordinated expression all genes of the gene cluster in the recipient cell. A method according to claim 1, characterized in that the expression of all genes of the gene cluster in the recipient cell by means of T ₇ RNA polymerase takes place. Method according to claim 1 or 2, characterized that linking the gene cassettes by homologous recombination he follows. Method according to one of claims 1 to 3, characterized in that the introduction of the recombinant Transposons are made in the recipient cells by conjugation. Gene cassette for cloning at least one gene cluster, wherein the gene cassette comprises the following elements: - at least a promoter sequence; and - At least one transposition element. Gene cassette according to claim 5, characterized in that the promoter sequence comprises the recognition and binding site for the T ₇ RNA polymerase. Gene cassette according to claim 5 or 6, characterized that the gene cassette additionally contains at least one transposase gene, preferably the tnp gene. Gene cassette according to one of claims 5 to 7, characterized in that the transposition element is an OE-L or an OE-R element. Gene cassette according to one of claims 5 to 8, characterized in that the gene cassette in addition comprises at least one reporter gene. Gene cassette according to one of claims 5 to 9, characterized in that the gene cassette in addition comprises at least one selection marker. Gene cassette according to one of claims 5 to 10, characterized in that the gene cassette comprises a self-promoter antibiotic resistance gene, a reporter gene without its own promoter, a transposition element and a promoter for a non-bacterial RNA polymerase, preferably the T ₇ RNA polymerase. Gene cassette according to one of claims 5 to 10, characterized in that the gene cassette is an antibiotic resistance gene with its own promoter, a reporter gene without its own promoter, a for a Transposase coding gene, a transposition element and a promoter for a non-bacterial RNA polymerase includes. Gene cassette according to claim 11 or 12, characterized in that the antibiotic resistance gene is a gene for tetracycline resistance (Tc ^R ) or for gentamycin resistance (Gm ^R ). Gene cassette according to claim 11, 12 or 13, characterized in that the reporter gene is a gene for kanamycin resistance (Km ^R ) or a gene coding for a fluorescent protein gene. Gene cassette according to one of claims 5 to 14, characterized in that the gene cassette additionally comprises at least one replication origin (oriT). Gene cassette according to one of claims 5 to 15, characterized in that the gene cassette in addition at least one operator sequence, preferably the lac operator, includes. Composition for cloning, integration and Expression of at least one gene cluster, wherein the composition at least two gene cassettes according to one of the claims 5 to 16. Vector for cloning, integration and expression at least one gene cluster, wherein the vector is at least one gene cassette according to any one of claims 5 to 16. Vector according to claim 18, characterized that the vector contains two or more gene cassettes according to one of the claims 5 to 16. Recombinant nucleic acid molecule, the at least one nucleotide sequence for the synthesis of at least one Polypeptide, wherein the nucleotide sequence selected is from the group consisting of: a) nucleotide sequence which the gene cassette according to any one of claims 5 to 16; b) Nucleotide sequence comprising two gene cassettes according to any one of claims 5 to 16; c) Nucleotide sequence following the vector Claim 18 or 19; d) nucleotide sequence which the Sequence according to SEQ ID NO. 13 and / or 14; e) Nucleotide sequence which differs from the nucleotide sequence according to a), b), c) or d) by the exchange of at least one codon against distinguishes a synonymous codon; f) nucleotide sequence whose complementary strand with the nucleotide sequences according to a), b), c) or d) hybridizes; g) nucleotide sequence which belongs to at least 85%, preferably 90%, especially 95%, with the nucleotide sequence according to a), b), c) or d) is identical; H) Nucleotide sequence corresponding to the complementary strand of the Nucleotide sequences according to a) to g) corresponds. Kit for cloning, integration and expression at least a gene cluster containing at least one gene cassette after a of claims 5 to 16. The kit of claim 21, which comprises two or more gene cassettes according to any one of claims 5 to 16. Cell containing at least one gene cassette after any one of claims 5 to 16, the vector of claim 18 or 19 and / or the nucleic acid molecule according to Claim 20 contains. Cell culture comprising cells according to claim 23.