MEANS FOR GENERATING ADENOVIRAL VECTORS FOR CLONING LARGE NUCLEIC ACIDS

Abstract

The present invention is related to a nucleic acid molecule, which is also referred to as third nucleic acid molecule, wherein the third nucleic acid molecule comprises (1) a nucleic acid molecule comprising the following elements: (a) optionally, a first part of a genome of a virus; (b) a nucleotide sequence, preferably a genomic nucleotide sequence, or a transcription unit; (c) a regulatory nucleic acid sequence which has a regulatory activity in a prokaryote; (d) exactly one site-specific recombination site; (e) a nucleotide sequence providing for a negative selection marker; (f) a bacterial nucleotide sequence unit comprising (i) bacterial nucleotide sequences for conditional replication and (ii) a nucleotide sequence providing for a positive selection marker; (g) optionally a first restriction site; or (2) a nucleic acid molecule comprising a nucleotide sequence according to SEQ ID NO: 6; or (3) a nucleic acid molecule identical or similar to the nucleic acid molecule contained in the organism deposited with the DSMZ under the Budapest treaty under accession number DSM 23754, wherein preferably the nucleic acid molecule contained in the organism is a heterologous nucleic acid molecule; wherein the third nucleic acid molecule is either a linear or a circular molecule.

Claims

1. A nucleic acid molecule, which is also referred to as third nucleic acid molecule, wherein the third nucleic acid molecule comprises (1) a nucleic acid molecule comprising the following elements: (a) optionally, a first part of a genome of a virus; (b) a nucleotide sequence, preferably a genomic nucleotide sequence, or a transcription unit; (c) a regulatory nucleic acid sequence which has a regulatory activity in a prokaryote; (d) a site-specific recombination site; (e) a nucleotide sequence providing for a negative selection marker; (f) a bacterial nucleotide sequence unit comprising (i) bacterial nucleotide sequences for conditional replication and (ii) a nucleotide sequence providing for a positive selection marker; and (g) optionally a first restriction site; or (2) a nucleic acid molecule comprising a nucleotide sequence according to SEQ ID NO: 6; or (3) a nucleic acid molecule identical or similar to the nucleic acid molecule contained in the organism deposited with the DSMZ under the Budapest treaty under accession number DSM 23754, wherein preferably the nucleic acid molecule contained in the organism is a heterologous nucleic acid molecule; wherein the third nucleic acid molecule is either a linear or a circular molecule.

2. The third nucleic acid molecule according to claim 1, wherein in the nucleic acid molecule of (1) the regulatory nucleic acid sequence which has a regulatory activity in a prokaryote, the site-specific recombination site and the nucleotide sequence providing for a negative selection marker are arranged in a 5 to 3 direction.

3. The third nucleic acid molecule according to any one of claims 1 to 2, wherein the third nucleic acid molecule contains exactly one site-specific recombination site.

4. The third nucleic acid molecule according to any one of claims 1 to 3, wherein the third nucleic acid molecule is a linear molecule, wherein elements (a) to (f), preferably upon cleavage of the circular molecule of the third nucleic acid molecule with the first restriction enzyme which recognized and cleaves at the first restriction site, are arranged in a 5 3 direction in the following sequence as follows: 1. optionally the first part of a genome of a virus; 2. the nucleotide sequence, preferably a genomic nucleotide sequence, or a transcription unit; 3. the regulatory nucleic acid sequence which has a regulatory activity in a prokaryote; 4. the site-specific recombination site; 5. the nucleotide sequence providing for a negative selection marker; and 6. the bacterial nucleotide sequence unit comprising (i) bacterial nucleotide sequences for conditional replication and (ii) a nucleotide sequence providing for a positive selection marker.

5. The third nucleic acid molecule according to any one of claims 1 to 4, wherein the third nucleic acid molecule further comprises a first part of a genome of a virus.

6. The third nucleic acid molecule according to claim 5, wherein the first part of a or the genome of a virus comprises the terminal sequence of a or the genome of a or the virus or one or several parts of the terminal sequence.

7. The third nucleic acid molecule according to any one of claims 5 to 6, wherein the first part of a or the genome of a or the virus is a first part of the genome of an adenovirus, preferably a human adenovirus and more preferably the adenovirus is human adenovirus type 5, and most preferably the entire left end of adenovirus type 5 upstream of the TATA box of the E1 transcription unit, or one or several parts thereof.

8. The third nucleic acid molecule according to any one of claims 1 to 7, preferably claim 7, wherein the bacterial nucleotide sequences for conditional replication comprise an origin of replication, whereby preferably the origin of replication is the minimal origin of phage gR6K.

9. The third nucleic acid molecule according to any one of claims 1 to 8, preferably any one of claims 7 to 8, wherein the regulatory sequence which has a regulatory activity in a prokaryote is a sequence which directs expression of a nucleotide sequence in a prokaryote, preferably in a prokaryotic host cell.

10. The third nucleic acid molecule according to any one of claims 1 to 9, preferably any of claims 8 to 9, wherein the negative selection marker or the expression of the nucleotide sequence providing for a negative selection marker mediates or confers sensitivity to a selecting agent and/or a selecting condition.

11. The third nucleic acid molecule according to claim 10, wherein the nucleotide sequence providing for a negative selection marker is a gene selected from the group comprising the galK, tetAR, pheS, thyA, lacy, ccdB and rpsL gene.

12. A combination of a third nucleic acid molecule as defined in any of claims 1 to 11 and a nucleic acid molecule which is also referred to as second nucleic acid molecule, wherein the second nucleic acid molecule comprises (1) a nucleic acid molecule comprising the following elements: (a) a bacterial nucleotide sequence unit comprising (i) bacterial nucleotide sequences for single copy replication, and (ii) a nucleotide sequence providing for a second selection marker; (b) a site-specific recombination site; (c) a second part of a genome of a virus; and (d) optionally a restriction site which is referred to as second restriction site; or (2) a nucleic acid molecule comprising a nucleotide sequence according to SEQ ID NO: 2 and/or SEQ ID NO: 13 and/or SEQ ID NO: 14; or (3) a nucleic acid molecule identical or similar to the nucleic acid molecule contained in the organism deposited with the DSMZ under the Budapest treaty under accession number DSM 24298 and/or DSM 24299, wherein preferably the nucleic acid molecule contained in the organism is a heterologous nucleic acid molecule; wherein the second nucleic acid molecule and the third nucleic acid molecule each and independently is either a linear molecule or a circular molecule; preferably the second nucleic acid molecule is a circular molecule and the third nucleic acid molecule is a circular molecule.

13. A combination of a nucleic acid molecule which is also referred to as first nucleic acid molecule, and a nucleic acid molecule which is also referred to as second nucleic acid molecule, wherein the first nucleic acid molecule comprises (1) a nucleic acid molecule comprising, the following elements: (a) a site-specific recombination site; (b) a bacterial nucleotide sequence unit comprising (i) bacterial nucleotide sequences for conditional replication and (ii) a nucleotide sequence providing for a first selection marker; (c) a first part of a genome of a virus; (d) a transcription unit; and (e) optionally a first restriction site; or (2) a nucleic acid molecule comprising a nucleotide sequence according to SEQ ID NO:1 and/or SEQ ID No:15; or (3) a nucleic acid molecule being similar or identical to the nucleic acid molecule contained in the organism deposited with the DSMZ according to the Budapest treaty under accession number DSM 23753, wherein preferably the nucleic acid molecule contained in the organism is a heterologous nucleic acid molecule; and wherein t second nucleic acid molecule comprises (1) a nucleic acid molecule comprising the following elements: (a) a bacterial nucleotide sequence unit comprising (i) bacterial nucleotide sequences for single copy replication, and (ii) a nucleotide sequence providing for a second selection marker; (b) a site-specific recombination site; (c) a second part of a genome of a virus; and (d) optionally a restriction site which is referred to as second restriction site; or (2) a nucleic acid molecule comprising a nucleotide sequence according to SEQ ID NO: 2 and/or SEQ IL) NO: 13 and/or SEQ ID NO: 14; or (3) a nucleic acid molecule identical or similar to the nucleic acid molecule contained in the organism deposited with the DSMZ under the Budapest treaty under accession number DSM 24298 and/or DSM 24299, wherein preferably the nucleic acid molecule contained in the organism is a heterologous nucleic acid molecule; and wherein the first nucleic acid molecule and the second nucleic acid molecule each and independently is either a linear molecule or a circular molecule, preferably the first nucleic acid molecule is a circular molecule and the second nucleic acid molecule a circular molecule.

14. The combination according to claim 13, wherein the first nucleic acid molecule contains exactly one site-specific recombination site.

15. The combination according to any one of claims 13 and 14, wherein the genome of a virus of the first nucleic acid molecule is a human adenovirus genome, preferably a human adenovirus genome which is different from human adenovirus type 5 genome, more preferably the genome of a virus of the first nucleic acid molecule is a human adenoviral type 19a genome.

16. The combination according to any one of claims 13 to 15, wherein the bacterial nucleotide sequences for conditional replication of the first nucleic acid molecule comprise an origin of replication.

17. The combination according to any one of claims 13 to 16, wherein the sequence providing for a first selection marker of the first nucleic acid molecule is a nucleic acid sequence coding for an enzyme which is conferring resistance to a host cell harbouring such nucleic acid sequence coding for an enzyme.

18. The combination according to any one of claims 13 to 17, wherein the first part of a genome of a virus of the first nucleic acid molecule is a viral terminal repeat, preferably an adenoviral terminal repeat.

19. The combination according to any one of claims 13 to 18, wherein the first part of a genome of a virus of the first nucleic acid molecule contains the adenoviral promoter pIX, more preferably the adenoviral promoter pIX is a pIX promoter from human adenovirus 19a.

20. The combination according to any one of claims 12 to 19, wherein the second nucleic acid molecule contains exactly one site-specific recombination site.

21. The combination according to any one of claims 12 to 20, wherein the virus genome of the second nucleic acid molecule is a human adenovirus genome, whereby in case of the combination according to claim 12 the virus genome of the second nucleic acid molecule is preferably a human adenovirus type 5 genome or a human adenoviral type 19a genome and in case of the combination according to claim 13 the virus genome of the second nucleic acid molecule is preferably a human adenovirus genome which is different from human adenovirus type 5 genome, more preferably the virus genome of the second nucleic acid molecule is a human adenoviral type 19a genome.

22. The combination according to any one of claims 12 to 21, wherein the bacterial nucleotide sequence for single copy replication of the second nucleic acid molecule comprises a replication origin for single copy maintenance in prokaryotic host cells.

23. The combination according to any one of claims 12 to 22, wherein the nucleotide sequence providing for a second selection marker of the second nucleic acid molecule marker is a nucleic acid sequence coding for an enzyme which is conferring resistance to a host cell harbouring such nucleic acid sequence coding to an enzyme.

24. The combination according to any one of claims 12 to 23, wherein the second part of a genome of a virus of the second nucleic acid molecule comprises an inverted terminal repeat of a virus, preferably an adenoviral inverted terminal repeat and more preferably an adenoviral right inverted terminal repeat.

25. A method for the generation of a nucleic acid molecule coding for a virus comprising the following steps a) providing a third nucleic acid molecule as defined in any one of claims 1 to 11; b) providing a second nucleic acid molecule as defined in claim 12; or c) a combination of a third nucleic acid molecule and a second nucleic acid molecule according to any one of claims 12 to 24; d) allowing the third and the second nucleic acid molecule to react so that a site-specific recombination occurs, wherein the site-specific recombination is mediated by a site-specific recombinase and the site-specific recombination forms a recombination product comprising a copy, preferably single copy of the genome of a or the virus, whereby the genome is a complemented complete genome and the complemented complete genome is complemented by the site-specific recombination; e) optionally selecting the recombination product; and f) optionally cleaving the recombination product with the first and second restriction enzyme.

26. A method for the generation of a nucleic acid molecule coding for a virus comprising the following steps a) a combination of a first nucleic acid molecule and a second nucleic acid molecule according to any one of claims 13 to 24; b) allowing the first and the second nucleic acid molecule to react so that a site-specific recombination occurs, wherein the site-specific recombination is mediated by a site-specific recombinase and the site-specific recombination forms a recombination product comprising a copy, preferably single copy of the genome of a or the virus, whereby the genome is a complemented complete genome and the complemented complete genome is complemented by the site-specific recombination; c) optionally selecting the recombination product; and d) optionally cleaving the recombination product with the first and second restriction enzyme.

27. The method according to claim 25, wherein the third and the second nucleic acid molecule are reacted in a prokaryotic host cell preferably E. coli, being similar or identical to the deposited organisms at the DSMZ with the accession numbers according to the Budapest treaty DSM 23743.

28. The method according to claim 26, wherein the first and the second nucleic acid molecule are reacted in a prokaryotic host cell preferably E. coli, being similar or identical to the deposited organisms at the DSMZ with the accession numbers according to the Budapest treaty DSM 23743.

29. A method for generating a library of nucleotide sequences, wherein said library comprises a plurality of individual nucleotide sequences, wherein said library is represented by a plurality of viral genomes and each viral genome contains a single one of the individual nucleotide sequences, comprising the steps of the method as defined in any of claims 25 and 27, wherein the individual nucleotide sequence is part of the transcription unit of the third nucleic acid molecule.

30. A method for generating a library of nucleotide sequences, wherein said library comprises a plurality of individual nucleotide sequences, wherein said library is represented by a plurality of viral genomes and each viral genome contains a single one of the individual nucleotide sequences, comprising the steps of the method as defined in any of claims 26 and 28, wherein the individual nucleotide sequence is part of the transcription unit of the first nucleic acid molecule.

31. A kit comprising optionally a package insert, and, in (a) suitable container(s), at least a third nucleic acid molecule as defined in any one of claims 1 to 11 and/or a combination of the third nucleic acid molecule and the second nucleic acid molecule according to any one of claims 12 to 24.

32. A kit comprising optionally a package insert, and, in (a) suitable container(s), at least a first nucleic acid molecule as defined in any one of claims 13 to 19 and/or a combination of the first nucleic acid molecule and the second nucleic acid molecule according to any one of claims 13 to 24.

33. The kit according to any one of claims 31 and 32, wherein the nucleic acid molecule(s) is/are contained in a ready-to-use form and/or wherein the kit contains instructions for use.

Description

[0493] The invention will now be described by reference to the following figures and examples which are merely illustrative and are not to be considered as a limitation of the scope of the invention.

[0494] FIG. 1 is a diagrammatic representation showing a method for constructing a first generation adenovirus genome, whereby a first nucleic acid identical or similar to pDonorSir1, and a second nucleic acid molecule identical or similar to pBACSir1 are combined and reacted through their recombination sites forming a fourth nucleic acid (pRAB) as recombination product which can be selected and contains exactly one copy of a complete complemented virus genome. Bacteria harboring the fourth nucleic acid containing the first and second selection marker and can be selected with the first and second selecting agent. The composition of a fourth nucleic acid molecule resulting from a single recombination event (pRAB1x) is given in FIG. 1A, the composition of a fourth nucleic acid molecule resulting from a double recombination event (pRAB2x) is given in FIG. 1B.

[0495] FIG. 2A illustrates the composition of DNA from recombinant adenovirus BACs analyzed by restriction digest with a restriction enzyme, and the composition of the DNA from two reconstituted complemented first generation adenovirus viruses generated from these BACs using the disclosed method of example 1 and example 2

[0496] FIGS. 2B-2M illustrate the composition of DNA from recombinant adenovirus BACs analyzed by restriction digest with a restriction enzyme obtained after site-specific recombination in E. coli using the disclosed method of example 3

[0497] FIG. 3 is a diagrammatic representation of the method disclosed in example 3 for constructing a plurality or library of fifth nucleic acid molecules. A third nucleic acid identical or similar to pDonorSir2, and a second nucleic identical or similar to pBACSir2 acid are combined and reacted through their recombination sites forming a fifth nucleic acid as recombination product which can be selected and contains exactly one copy of a complete complemented virus genome (FIG. 3A). Bacteria harboring the fifth nucleic acid containing the positive, the negative selection marker from the third nucleic acid, and the second selection marker, can be selected. The schematic composition of a fifth nucleic acid molecule resulting from a single recombination event (pRAB_RPSL1x) is given in FIG. 3A, the schematic composition of a fifth nucleic acid molecule resulting from a double recombination event ((pRAB_RPSL2x)) is given in FIG. 3B.

[0498] FIG. 4 shows GFP expressing adenovirus vectors obtained after direct transfection of linearized forms of the first and the second nucleic acid in 293 cells expressing the site-specific recombinase Flp using the disclosed method of example 5.

[0499] FIG. 5 illustrates the selective inhibition of growth of bacteria in medium containing the negative selecting agent at different concentrations harboring a double recombined BAC (pRAB_RPSL2x) according to the disclosed method of example 3.

[0500] FIG. 6 shows the combinations of positive and negative selection marker useful for generation of a plurality of fifth nucleic acid molecules according to the method provided by this invention.

[0501] FIG. 7 illustrates the composition of DNA from recombinant human adenovirus type 19a BACs analyzed by restriction digest with a restriction enzyme using the disclosed method of example 6

BRIEF DESCRIPTION OF THE FIGURES

[0502] FIG. 1 Diagrammatic representation showing a method for constructing a first generation adenovirus genome, whereby two nucleic acids are combined and reacted through their recombination sites forming a recombination product corresponding to a fourth nucleic acid molecule according to a disclosed method of example 1 and 2. The fourth nucleic acid generated can be selected and contains exactly one copy of a complete complemented virus genome. The reaction product can be cleaved optionally with a first and a second restriction enzyme in order to release a complete virus genome that can be replicated in a permissive cell. According to example 1 a first nucleic acid vector identical or similar to pDonorSir1 (Seq. ID. No. 1) contains a minimal Frt34 recombination site (SEQ. ID. No. 7) derived from the wild type Frt site, a bacterial nucleotide sequence comprising (i) bacterial nucleotide sequences for conditional replication (OriR6K) and a nucleotide sequence providing for a first selection marker conferring a host cell resistance against kanamycin (KnR), a first restriction site (RS1), a first part of a genome of a virus containing the left ITR of an adenovirus genome (ITRleft) and the packaging signal ES, and a transcription unit (TU). The second nucleic acid is a BAC vector identical or similar to pBACSir1 (Seq. ID NO. 13) or pBACSir2 (Seq. ID No.2), comprising a wild type Frt48 recombination (SEQ. ID. No. 8) site, a second part of the genome of an adenovirus (Ad) comprising the right ITR of an adenovirus genome (ITRrigth), a second restriction site (RS2), and a bacterial nucleotide sequence unit comprising (i) bacterial nucleotide sequences for single copy replication (F'ori), and (ii) a nucleotide sequence providing for a second selection marker conferring a host cell resistance against chloramphenicol (CmR). Both nucleic acid molecules are reacted through their recombination sites in a bacterial host cell in the presence of Flp recombinase. The resulting fourth nucleic acid molecules are recombinant adenovirus BACs (pRABs) either consisting of single recombined products, or multiple recombined products. In FIG. 1A the schematic composition of a single recombination product (pRAB1x) (Seq. ID. No. 4) is given. In FIG. 1B the schematic composition of a double (pRAB2x) recombination product (Seq. ID. No. 5) is given. Upon digestion of the DNA of a fourth nucleic acid with the first and the second restriction enzyme, a complete adenovirus genome is released from the pRABs containing the left and the right ITR, the packaging signal, and the transcription unit (FIG. 1C). Viable first generation adenovirus vectors are obtained in 97% of the cases if the DNA of a fourth nucleic acid obtained according to the disclosed method in example 1 and digested with the first and second restriction enzyme according to the disclosed method in example 2 is transfected into permissive 293 cells.

[0503] FIG. 2A illustrates the composition of two reconstituted first generation adenovirus viruses obtained using the method disclosed in example 1. The DNA of two types of reaction products are pRAB1x and pRAB2x, resulting from single and double insertion of pDonorSir1 into pBACSir1, whereby pDonorSir1 is identical to the deposited organism at the DSMZ according to the Budapest treaty with the accession number DSM 23753, and whereby pBAcSir1 is identical to the deposited organism at the DSMZ according to the Budapest treaty with the accession number DSM 24298. The respective reaction products were isolated from a growing culture of DH10B bacteria according to standard protocols, and characterized by restriction digest with XhoI (FIG. 2A, lanes 3-4). In lane 1 a nucleotide length marker was loaded, providing reference fragments with defined length between 1 and 10 kb. In lane 2 the restriction pattern of the recombinant adenovirus BAC vector pRABref (Seq. ID. No. 3) is given as a reference. The in silico generated XhoI restriction pattern of pRABref is as follows: 14.5 kb, 10.274 kb, 7.403 kb, 2.466 kb, 1.445 kb, and 0.595 kb. Analysis of the single recombination product RAB1x (lane 3) yields a characteristic additional pair of bands of 6.266 kb and 4.187 kb length, respectively. The in silico generated pattern for digestion with XhoI of the single recombined reaction product RAB1x (Seq. ID. No. 4) is as follows: 14.5 kb, 10.274 kb, 6.266 kb, 4.187 kb, 2.466 kb, 1.445 kb, and 0.595 kb. In case of the double recombined product, a third additional band of 3.05 kb appears in the (lane 4). The in silico generated pattern for digestion with XhoI of the double recombined reaction product RAB2x (Seq. ID. No. 5) is as follows: 14.5 kb, 10.274 kb, 6.266 kb, 4.187 kb, 3.05 kb, 2.466 kb, 1.445 kb, and 0.595 kb. In a further experiment the RAB1x and RAB2DNA was isolated and cut with PacI restriction enzymes (corresponding to RS1 and RS2 in FIG. 1, respectively), and transfected into permissive 293 cells. FIG. 2A shows the restriction pattern of virus DNA isolated from 293 cells transfected with PacI-restricted RAB1x (lane 6) and RAB2x (lane 7). The in silico generated pattern for digestion with XhoI of the viral DNA obtained from both reconstituted recombinant adenoviruses, RAB1x and RAB2x respectively, is as follows: 14.5 kb, 8.499 kb, 3.365 kb, 2.466 kb, 1.445 kb, and 0.595 kb. The restriction fragment pattern is identical for both viruses since the identical complete complemented adenovirus genome is liberated from the pRABs upon digestion with PacI. The restriction pattern of RAB1x or RAB2x was compared to an empty adenovirus, being essentially the same as RAB1x and RAB2x, but lacking the transcription unit (FIG. 2, lane 5). The in silico restriction fragment pattern upon digestion with XhoI of the adenovirus DNA isolated form RAB1x or RAB2x, respectively, is as follows: 14.5 kb, 8.499 kb, 3.365 kb, 2.466 kb, 1.445 kb, and 0.595 kb.

[0504] FIGS. 2B-2M show the restriction fragment analysis of the recombination products between pDonorSir2 and pBACSir2 obtained according the method disclosed in example 3, whereby pDonorSir2 is identical to the deposited organism at the DSMZ according to the Budapest treaty with the accession number DSM 23754, and whereby pBACSir2 is identical to the deposited organism at the DSMZ with the accession number according to the Budapest treaty DSM 24299. Selection of recombination products took place onto agar plates which contained kanamycin (25 g/ml) chloramphenicol (25 g/ml), and streptomycin sulphate (50 g/ml) as selecting agents. Under these conditions E. coli contained recombined recombinant adenovirus BACs (pRABs), and contained pRAB_RPSL_1x (Seq. ID. No. 11) as reaction product in 83 out of 88 analyzed reaction products. In only 2 out of 88 cases a double recombined reaction product pRAB_RPSL_2x (Seq. ID. No. 12) was obtained. Single colonies were picked from the selection plate, subcultured in liquid media containing chloramphenicol (25 g/ml) and BAC DNA from subsequent subcultures of the colonies containing pRABs was isolated and the integrity of the reaction products analyzed by restriction fragment analysis upon digestion with XhoI (FIGS. 2B-2M). All the 6 analysed recombinants analyzed, contained pRAB_RPSL_1x. To test the reliability of the method, the experiment was repeated and a further 82 clones were picked from the selection plates and characterized as above. We could find only 2 clones which contained multiple insertion products corresponding to pRAB_RPSL_2x marked with D in FIG. 2F; clones number #47 and #53, respectively. Further 7 BAC DNA preparations were contaminated by the parental vector pBACSir2 (marked with V, clone number #9,#17,#22,#39,#41,#62,#67), and 3 were recombination products resulted from rearrangements (marked with r, clone number #25,#46,#68. In a further experiment a total of 44BACs corresponding to a fifth nucleic acid were analyzed, whereby only 1 recombination product corresponded to pRAB_RPSL_2x and 43 of the 44 clones corresponded to pRAB_RPSL_1x. Altogether 126/132 BACs corresponded to the single recombination product pRAB_RPSL_1x (95.5% of the recombination products analyzed) and multiple recombination was observed in 3/132 corresponding to 2.3% of the clones.

[0505] FIG. 3 Diagrammatic representation showing the method disclosed in example 3 for constructing complemented complete adenovirus vector genomes, or a plurality or library of those. The recombination between a third nucleic acid molecule and a second nucleic acid molecule are combined and reacted through their Frt recombination sites forming a recombination product which can be selected, and whereby the number of recombination events is limited to one. According to example 3 a third nucleic acid molecule identical or similar to pDonorSir2 containing a prokaryotic promoter (PK promoter), a minimal Frt34 recombination site derived from the wild type Frt site, a negative selection marker (Rps1), a bacterial nucleotide sequence comprising (i) bacterial nucleotide sequences for conditional replication (OriR6K) and a nucleotide sequence providing for a positive selection marker conferring a host cell resistance to kanamycin (KnR), a first restriction site (RS1), a first part of a genome of a virus containing the left ITR of an adenovirus genome (ITRleft) and the packaging signal ES, a transcription unit or gene of interest (GOI). The second nucleic acid, which is identical or similar to pBACSir2 (SEQ. ID. No. 2) comprises a wild type Frt48 recombination site (SEQ. ID. No. 8), a second part of the genome of an adenovirus comprising the right ITR of an adenovirus genome (ITRrigth), a second restriction site (RS2), and a bacterial nucleotide sequence unit comprising (i) bacterial nucleotide sequences for single copy replication (F'ori), and (ii) a nucleotide sequence providing for a second selection marker conferring a host cell resistance to chloramphenicol (CmR). Both nucleic acid molecules are reacted through their recombination sites in a bacterial host cell in the presence of Flp recombinase. Using the method disclosed, the resulting recombinant BACs predominantly (>95%) consist of single recombined products. In FIG. 3A the schematic composition of a single recombination product pRAB_RPSL_1x is given. In FIG. 3B the double recombination is depicted schematically. In the recombination product pRAB_RPSL_2x the prokaryotic promoter (PK Promoter) is in proximity to the open reading frame of the negative selection marker (Rps1). This product is observed in less than 2.5% of the recombination products according to the disclosed method in example 3.

[0506] FIG. 4 shows the reconstitution of complemented infectious adenovirus viruses in 293 Flp cells expressing Flp recombinase according to a disclosed method in example 5, whereby 293 Flp cells are identical to the deposited organism at the DSMZ with the accession number according to the Budapest treaty DSM ACC3077. 293Flp cells were transfected with a first nucleic acid molecule corresponding to pSirDonor1-EGFP (SEQ. ID No.9), and a second nucleic acid molecule corresponding to pBACSir2, whereby both nucleic acids were treated with PacI prior to transfection. After 3 days cultivation at 37 C. under standard cell culture conditions comet-shaped fluorescent conglomerates of cells showing cytopathic effect (CPE) characteristic for productive adenovirus production in 293 cells were microscopically detected.

[0507] FIG. 5 shows the selective inhibition of the growth by streptomycin of E. coli DH10B cells carrying pRAB_RPSL_2x obtained from double recombination of pDonorSir2 with pBACSir2. Growth curves were generated from E. coli DH10B cells transformed with the empty vector pBACSir2, the single recombined pRAB_RPSL_1x_#1, or two double recombined adenovirus BAC vectors pRAB_RPSL_2x_#47, and pRAB_RPSL_2x_#53, respectively. Growth of bacterial cultures was done at different concentrations of streptomycin starting from a diluted saturated overnight culture as starting material, and monitoring of the OD600 over time. After the average of the OD600 of the replicate culture containing the control BAC vector pBACSir2 reached 0.8, usually 8 hours post inoculation, the OD600 values were measured and the optical density calculated and referenced to the average OD600 of the control culture which was set to 100%. The results of 5 independent experiments were plotted for each growth conditions and standard deviations of the relative optical densities within the plotted 5 experiments included as error bars. The E. coli clones carrying the control BAC vector pBACSir2 or the single recombined BAC vector pRAB_RPSL_1x grew well even in the presence of very high concentration (200 g/ml) of streptomycin. In contrast the growth of the clones carrying a double insertion of pDonorSir2 (pRAB_RPSL_2x #47) and pRAB_RPSL_2x_#53) was blocked by 50 g/ml streptomycin, some inhibition was detectable already in the presence of 25 g/ml streptomycin.

[0508] FIG. 6 shows a synergy matrix of selection markers. Based on the mode of action of antibiotics, positive and negative selection markers have the potential to work synergistically in the presence of kanamycin. Combinations of positive and negative selection markers that are expected to work synergistically for the counter-selection of multiple-recombined products according to the method of the present invention described herein for the generation of recombinant adenovirus virus vectors are marked + in the table, and combinations that are not expected to work synergistically are marked with 0.

[0509] FIG. 7 illustrates the composition of two recombinant first generation adenovirus type 19a vectors obtained using the method disclosed in example 6. The reaction products were isolated from a growing culture of DH10B bacteria according to standard protocols, and characterized by restriction digest with KpnI. In the first lane marked with M a nucleotide length marker was loaded, providing reference DNA fragments with defined length between 1 and 10 kb. Restriction analysis with KpnI of the single recombination product pRAB19a1x (lane 1,2,3) and double recombination products (lanes 4) are shown. The in silico generated pattern for digestion with KpnI of the single recombined reaction product pRAB19a1x (Seq. ID. No. 16) is as follows: 11.361 kb, 6.254 kb, 5.447 kb, 4.443 kb, 3.271 kb, 2.016 kb, 1.886 kb, 1.868 kb, 1.585 kb, and 28 bp. In case of the double recombined product, an additional band of 3.364 kb appears (lane 4). The in silico generated pattern for digestion with KpnI of the double recombined reaction product pRAB19a2x (Seq. ID. No. 17) is as follows: 11.361 kb, 6.254 kb, 5.447 kb, 4.443 kb, 3.364 kb, 3.271 kb, 2.016 kb, 1.886 kb, 1.868 kb, 1.585 kb, and 28 bp.

EXAMPLES

Example 1: Construction of Recombinant Adenovirus BACs Using Site-Specific Recombination in E. coli Expressing Flp Recombinase

[0510] For construction of a recombinant adenovirus genome, a first nucleic acid pDonorSir1 and a second nucleic acid molecule pBACSir1 were combined and reacted in DH10B E. coli cells harbouring pBACSir1 and the plasmid pCP20 for conditional expression of FLP recombinase, whereby pDonorSir1 is identical to the deposited organism at the DSMZ with the accession number according to the Budapest treaty DSM 23753, and whereby pBACSir1 is identical to the deposited organism at the DSMZ according to the Budapest treaty with the accession number DSM24298, and whereby E. coli cells harbouring pBACSir1 and pCP20 are identical to the deposited organism at the DSMZ according to the Budapest treaty with the accession number DSM 23742. The plasmid pDonorSir1 was introduced into the DH10B E. coli cells by means of electroporation using a standard protocol. The nucleic acid molecule pBACSir1 is a derivative of the pKSO BAC vector (Messerle et al. Proc. Natl. Acad. Sci. U.S.A 94:14759-14763, 1997) and contain the right part of the human adenovirus type 5 (AV5) genome deleted for the E1 region and the E3 region. The nucleic acid molecule pBACSir1 was maintained in E. coli DH10B (or equivalent E. coli K12-derived strains lacking the F factor) harbouring a conditional expression system for expression of Flp. Here, in example 1, the DH10B cells harboured the adenovirus BAC pBACSir1, and the Flp recombinase was provided by the plasmid pCP20, which replication is controlled by a temperature-sensitive origin of replication (Bubeck A. et al., J. Virol. 78:8026-8035, 2004). DH10B cells harbouring pBACSir1 and the pCP20 were maintained at 30 C. in the presence of ampicillin (50 g/ml) and chloramphenicol (25 g/ml). Next, these DH10B cells were electro-transformed with pDonorSir1 and cultured for 60 minutes at 42 C. in the absence of any antibiotics. The expressed Flp induced site-specific recombination between FRT sites present on pDonorSir1 and pBACSir1, respectively. At the same time the elimination of Flp expression also started, since pCP20 cannot replicate in E. coli at elevated temperature. The transformed culture was plated onto agar plates which contained kanamycin (25 g/ml) and chloramphenicol (25 g/ml) as selecting agents. Under these conditions E. coli containing recombined recombinant adenovirus BACs (pRABs) were selected in which at least one pDonorSir1 plasmid had recombined with pBACSir1. DNA from growing cultures of DH10B cells containing pRABs was isolated and the integrity of the reaction products analyzed by restriction digestion with XhoI (FIG. 2A (lanes 2-4). All the recombination products analyzed contained pRABs, either being single (pRAB1x) or multiply recombined products (pRAB2x).

Example 2: Reconstitution of Recombinant Adenoviruses Generated by Site-Specific Recombination in E. coli Expressing Flp Recombinase

[0511] The two predominant types of BAC vectors obtained from site-specific recombination according to the disclosed method in example 1 were pRAB1x and pRAB2x, respectively. The pRABs generated by the Flp-recombination in DH10B cells contained one, and only one continuous sequences of a complete complemented adenovirus genome, which was replication competent in 293 cells. The DNA of pRABs was purified from saturated E. coli over night cultures (100 ml) in LB medium using a kit for plasmid preparation. Here, the Nucleobond PC-100 kit from Macherey and Nagel, Germany was used according to the manufacturer's recommendations. The identity of the pRBAs obtained was verified by means of restriction analysis of the pRAB DNAs (FIG. 2A, lanes 2-4). For virus reconstitution purified pRAB DNA was treated with 10 U PacI per g DNA for 2 h according to the manufacture's recommendations. Subsequently the PacI-digested pRAB1x and pRAB2x DNAs were purified using phenol-chloroform according to standard protocols prior to transfection into 293 cells. In brief, 10 g pRAB DNA was digested in a volume of 100 l for 1.5 h at 37 C. in a water bath. Subsequently 50 l phenol/chloroform (1:1 mixture) was added to the reaction tube (Eppendorff cup size 1.5 ml, Eppendorf AG, Hamburg, Germany) and vortexed for 20 sec. here, the Vortexer MS-3 basic was used (IKAIKA Werke GmbH & Co. KG, Staufen, Germany). The tube was centrifuged in a table top centrifuge at maximum speed (20000g) for 5 min at room temperature and 80 l of the aqueous upper phase was transfered into a fresh tube and 10 l 3 M NaAc (pH 4,5) and 200 jai EtOH was added. All reagents and chemicals were purchased from Sigma-Aldrich, St Louis, USA. The tube was mixed with the finger tips until the precipitated DNA became visible. Moreover, the tube was incubated for 5 min at room temperature and the DNA was pelleted in a table top centrifuge at maximum speed for 15 min at room temperature. The supernatant was quantitatively removed and the pellet immediately dissolved in 20 l sterile deionized water. Transfection of 293 cells was done using lipofection. Here, the Superfect transfection reagent (Qiagen, Hilden, Germany) was used according to the manufacturer's recommendation. The resulting adenoviruses were replication competent in 293 cells and could be propagated according to standard protocols (Green M and Loewenstein P, Human Adenoviruses: Propagation, Purification, Quantification, and Storage in Current Protocols in Microbiology.sub.79). The identity of the recombinant adenovirus vectors obtained according to the disclosed method in this example was verified by restriction digest of adenovirus vector DNA with XhoI and analysis of DNA fragments using agarose gel electrophoresis (FIG. 2A). For preparation of genomic adenovirus vector DNA, 293 cells (2,510.sub.7 cells) were infected with a MOI of 3 with the recombinant adenovirus vectors obtained after transfection of the PacI-digested pRABs into the 293 cells. After the cytopathic effect (CPE) was complete the infected cells were washed once in PBS, scraped from the plates and resuspent in PBS. Cells (4106 cells/ml) were lysed by adding an equal volume of TST buffer (2% TritonX-100, 400 mM NaCl, 20 mM Tris-HCl pH8.0) to the cell suspension followed by incubation on ice for 30 minutes. Cell debris were removed by centrifugation at 20,000 g for 10 minutes at 4 C. and the supernatant was treated with 50 g/ml proteinase K (Roche) in the presence of 0.5% SDS for 60 minutes at 56 C. After extraction of the nucleic acids by phenol/chloroform and ethanol precipitation the extract was treated with RNase A (Sigma). RNA-free viral DNA was again phenol/chloroform extracted and precipitated with ethanol. The XhoI restriction pattern of reconstituted virus derived from pRAB1x and pRAB2x corresponded to the in silico generated pattern, confirming the integrity of the adenovirus genome in the recombinant adenovirus viruses obtained (FIG. 2A lanes 5-7).

Example 3: Generation of Recombinant RABs with Controlled Recombination Through Negative Selection

[0512] To avoid multiple insertions and improve the construction of an adenovirus expression library, we constructed pDonorSir2 which is an embodiment of the third nucleic acid molecule of the present invention, whereby pDonorSir2 is identical to the deposited organism at the DSMZ according to the Budapest treaty with the accession number DSM 23754. pDonorSir2 differs from pDonorSir1 at its FRT locus, next to this pDonorSir2 contains a strong E. coli galaktokinase promoter (Warming, S., N. Costantino, Court D L, N. A. Jenkins, and N. G. Copeland. Simple and highly efficient BAC recombineering using galK selection. Nucleic Acids Res 2005, 33:e36) upstream to the FRT site and downstream of the FRT site a rpsL open reading frame, which mediated Streptomycin sensitivity if expressed (Reyrat J M, Pelicic V, Gicquel B, Rappuoli R. Counterselectable markers: untapped tools for bacterial genetics and pathogenesis. Infect. Immun 1998, 66:4011-4017). The use of pDonorSir2 is exemplified as follows: DH10B cells harbouring pBACSir2 and pCP20 were maintained at 30 C. in the presence of ampicillin and chloramphenicol, whereby pBACSir2, which is an embodiment of the second nucleic acid molecule of the present invention, is identical to the deposited organism at the DSMZ with the accession number according to the Budapest treaty DSM 24299, and whereby E. coli cells harbouring pBACSir2 and pCP20 are identical to the deposited organism at the DSMZ according to the Budapest treaty with the accession number DSM 23743. Next, the DH10B cells were electro-transformed with pDonorSir2 and cultured for 150 minutes at 42 C. in the absence of any antibiotics. The expressed Flp induced site-specific recombination between FRT sites present on pDonorSir2 and pBACSir2, respectively. At the same time the elimination of Flp expression also started, since pCP20 cannot replicate in E. coli at elevated temperature. The transformed culture was plated onto agar plates which contained kanamycin (25 g/ml) chloramphenicol (25 g/ml) and streptomycin sulphate (50 g/ml) as selecting agents. Under these conditions E. coli containing recombined recombinant adenovirus BACs (pRAB_RPSL) were selected, in which the pDonorSir2 plasmid had recombined with pBACSir2. Single colonies were picked from the selection plate, and cultured in 10 ml liquid LB media containing chloramphenicol (25 g/ml) over night at 37 C. in a shaking incubator. All chemicals and media used were purchased from Sigma-Aldrich, St Louis, USA.

[0513] pRAB_RPSL DNA from these cultures was subsequently isolated according to the manufacture's recommendations using a DNA-plasmid isolation kit, and the integrity of the reaction products analyzed by restriction digestion with XhoI (FIGS. 2B-2M). Here, the Nucleobond PC-100 kit from Macherey and Nagel, Germany was used for isolation of pRAB_RPSL-DNA according to the manufacturer's recommendations. The XhoI restriction pattern of all 6 pRABs analysed corresponded to single recombined products (pRAB_RPSL_1x). To test the reliability of the applied counter selection we picked further 82 clones from the selection plates ant tested as above. Only 2 clones contained multiple insertion products (marked by D in FIG. 2E), further 7 clones were contaminated by pBACSir2 (marked by V in FIG. 2B FIGS. 2C, 2D, 2E, 2G, and 2H), and 3 contained other unidentified rearrangements (marked by r in FIGS. 2D, 2F, and 2H). Altogether the great majority (83/88) of the colonies contained only pRAB_RPSL_1x (FIGS. 2B-2M). In a further experiment a total of 44_clones were analyzed were analyzed, whereby only 1 recombination product corresponded to pRAB_RPSL_2x, and 43 of the 44 clones corresponded to pRAB_RPSL_1x. Altogether 126/132 BACs corresponded to the single recombination product pRAB_RPSL_1x (95.45% of the recombination products analyzed) and multiple recombination was observed in 3/132 corresponding to 2.3% of the clones.

Example 4: Determination of the Average Library Efficiency for Generation of Recombinant Adenovirus BAC Libraries

[0514] To test the efficiency of our E. coli recombination system and avoid the contamination of pRAB_RPSL DNA preparations according to example 3 with non-recombined pBACSir2 vector, the experiment described in Example 3 was repeated two more times with the following modifications:

i) To test the primary cloning efficiency we took 50 l of a 10 ml post-transformation culture and serial 10-fold dilutions were plated on a triple selection agarose plate containing kanamycin (25 g/ml), chloramphenicol (25 g/ml), and streptomycin sulphate (50 g/ml) as selecting agents (Experiment 2). All chemicals and media used were purchased from Sigma-Aldrich, St Louis, USA. After 60 minutes the rest of the culture was incubated for another 90 minutes giving finally 150 minutes total post-transformation culture time as above (Experiment 3). Two plates were inoculated by 200 l out of 1 ml final volume of each dilution of the 50 l post-transfection culture (1:10.sup.1, 1:10.sup.2, and 1:10.sup.3) from each experiment.
ii) After the colonies containing the pRABs appeared on selection plates we made replica plates on a second round of triple selection plates containing kanamycin (25 g/ml), chloramphenicol (25 g/ml), and streptomycin sulphate (50 g/ml) as selecting agents. This procedure minimized the contamination by both the vector and the multiple insertion products, whereby the generation of replica plates is applied regularly in maintaining E. coli libraries.

[0515] The colony counts on replica plates were 52 and 89 in experiment 2, and 204 and 129 in experiment 3 with the dilution 1:10.sup.2. Taking in account that 50 ng DNA of pDonorSir2 was used, and the volume of the post-transformation culture was 50 l , and one fifth of each dilution was plated, the average cloning efficiency was 1,8510.sup.6 colony for 1 g input.

Example 5: Generation of replication competent adenovirus in 293 cells expressing FLP Recombinase

[0516] For construction of HEK 293 Hp cells expressing Flp recombinase 2.510.sup.5 HEK 293 cells were transfected using lipofection with 10 g of the plasmid pFlp-Puro linearized with PvuI, whereby 293 Flp cells are identical to the deposited organism at the DSMZ according to the Budapest treaty with the accession number DSM ACC3077. Here, the Superfect transfection reagent (Qiagen, Hilden, Germany) was used according to the manufacturer's recommendation. The transfected cells were incubated for 48 h at 37 C. under standard cell culture conditions (95% humidity, 5% CO2). The cell culture medium used was DMEM containing 10% FCS, 2 mM Glutamin, and 1% penicillin/streptavidin (P/S)). For selection puromycin was added to a final concentration of 1 g/l to the medium, and cells were cultivated under selective conditions for 12 days to obtain 293 cells stably expressing FLP recombinase. All chemicals and media used were purchased from Sigma-Aldrich, St Louis, USA. The stable cell pool was expanded and a master cell bank established. For reconstitution of recombinant adenovirus 210.sup.5 293 FlpP cells per well were plated onto a 6 well plate and 5 hours after plating cells were co-transfected with 0.8 g pDonorSir2-EGFP (SEQ. ID No. 9) and 2.5 g pBACSir2, both linearized with PacI, using Lipofection. Here, the Superfect transfection reagent (Qiagen, Hilden, Germany) was used according to the manufacturer's recommendation. Following a 3 days cultivation at 37 C. under standard cell culture conditions, cells were harvested by scraping and collected by subsequent centrifugation for 5 min at 200g. Cell pellets were resuspent in 400 l cell culture medium (DMEM, 10% FCS, 2 mM Glutamin, 1% P/S) and subjected to three successive freeze/thaw cycles. Cell debris was separated from soluble material by centrifugation at 4.400g for 15 min. In order to demonstrate a successful rescue of adenovirus vectors expressing the EGFP gene, 210.sup.5 HEK-293 cells/well were plated onto a 6 well plate and infected 12 h later with 200 l of the freeze/thaw lysate followed by 3 days incubation at 37 C. under standard cell culture conditions. At this time point comet-shaped fluorescent conglomerates of cells showing cytopathic effect (CPE) characteristic for productive adenovirus replication were microscopically detectable (see FIG. 4). The method thus allowed for the generation of first generation recombinant replication competent adenovirus vectors by co-transfection of a third nucleic acid molecule with a second nucleic acid molecule into 293 Flp cells.

Example 6: Construction of Recombinant Adenovirus Type 19a BACs Using Site-Specific Recombination in E. coli Expressing Flp Recombinase

[0517] For construction of a human non-type 5 recombinant adenovirus genome, a first Ad19a nucleic acid pDonorSir19a, which is an embodiment of the first nucleic acid molecule of the present invention, and a second Ad19a nucleic acid molecule pBACSir19a, which is an embodiment of the second nucleic acid molecule of the present invention, were combined and reacted in DH10B E. coli cells harbouring pBACSir19a and the plasmid pCP20 for conditional expression of FLP recombinase. The plasmid pDonorSir19a was introduced into the DH10B E. coli cells by means of electroporation using a standard protocol. The nucleic acid Ad19a molecule pBACSir19a was maintained in E. coli DH10B (or equivalent E. coli K12-derived strains lacking the F factor) harbouring a conditional expression system for Flp. Here, in example 6, the DH10B cells harboured the adenovirus type 19a BAC pBACSir19a, and the Flp recombinase was provided by the plasmid pCP20, which replication is controlled by a temperature-sensitive origin of replication. DH10B cells harbouring pBACSir19a and the pCP20 were maintained at 30 C. in the presence of ampicillin (50 g/ml) and chloramphenicol (25 g/ml). Next, these DH10B cells were electro-transformed with pDonorSir19a and cultured for 60 minutes at 42 C. in the absence of any antibiotics. The expressed Flp induced site-specific recombination between FRT sites present on pDonorSir19a and pBACSir19a, respectively. At the same time the elimination of Flp expression also started, since pCP20 cannot replicate in E. coli at elevated temperature. The transformed culture was plated onto agar plates which contained kanamycin (25 g/ml) and chloramphenicol (25 g/ml) as selecting agents. Under these conditions E. coli containing recombined recombinant adenovirus type 19a BACs (pRAB19a) were selected in which at least one pDonorSir19a plasmid had recombined with pBACSir19a. DNA from growing cultures of DH10B cells containing pRAB19a's was isolated and the integrity of the reaction products analyzed by restriction digestion with KpnI (FIG. 7). All the recombination products analyzed contained pRAB19a's, either being single (pRAB19a1x Seq ID. No.16) or multiple recombined products (pRAB19a2x, Seq ID No.17).

Example 7: Generation of Human Non-Adenovirus Type 5 Recombinant RABs with Controlled Recombination Through Negative Selection

[0518] For construction of a plurality or library of human non-type 5 recombinant adenovirus genomes, a third Ad19a nucleic acid pDonorSir2_19 a, which is an embodiment of the third nucleic acid molecule of the present invention, and a second Ad19a nucleic acid molecule pBACSir19a, which is an embodiment of the second nucleic acid molecule of the present invention, are combined and reacted in DH10B E. coli cells harbouring pBACSir19a and the plasmid pCP20 for conditional expression of FLP recombinase. The plasmid pDonorSir2_Ad19a differs from pDonorSir2 at its FRT locus, next to this pDonorSir2_Ad19a contains a strong E. coli galaktokinase promoter (Warming S N et al. Nucleic Acids Res 2005, 33:e36) upstream to the FRT site and downstream of the FRT site a rpsL open reading frame, which mediated Streptomycin sensitivity if expressed (Reyrat J M et al. Infect. Immun 1998, 66:4011-4017). The donor nucleic acid pDonorSir2_19 a carries a PacI site, Ad19a ITR and packaging signal.

[0519] The use of pDonorSir2_Ad19a is exemplified as follows: DH10B cells harbouring pBACSir19a and pCP20 are maintained at 30 C. in the presence of ampicillin and chloramphenicol. Next, the DH10B cells are electro-transformed with pDonorSir2_Ad19a and cultured for 150 minutes at 42 C. in the absence of any antibiotics. The expressed Flp induces site-specific recombination between FRT sites present on pDonorSir2_Ad19a and pBACSir19a, respectively. At the same time the elimination of Flp expression starts, since pCP20 cannot replicate in E. coli at elevated temperature. The transformed culture is plated onto agar plates which contain kanamycin (25 g/ml) chloramphenicol (25 g/ml) and streptomycin sulphate (50 g/ml) as selecting agents. Under these conditions E. coli containing recombined recombinant adenovirus BACs are selected, in which the pDonorSir2_Ad19a plasmid has recombined with pBACSir19a. Single colonies are picked from the selection plate, and cultured in 10 ml liquid LB media containing chloramphenicol (25 g/ml) over night at 37 C. in a shaking incubator. All chemicals and media used are purchased from Sigma-Aldrich, St Louis, USA. DNA from recombination products from these cultures is subsequently isolated according to the manufacture's recommendations using a DNA-plasmid isolation kit, and the integrity of the reaction products analyzed by restriction digestion with KpnI. Here, the Nucleobond PC-100 kit from Macherey and Nagel, Germany is used. The KpnI restriction pattern corresponds to single recombined products.

Biological Material

[0520] The invention uses and/or relates to biological material deposited under the Budapest Treaty. More specifically, the following depositions have been made with Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ), also referred to herein as DSMZ: DSM 23753; DSM 24298; DSM 24299; DSM 23743; DSM 23742; DSM ACC3077m; DSM ACC3077; and DSM 23754.

[0521] The features of the present invention disclosed in the specification, the claims and/or the drawings may both separately and in any combination thereof be material for realizing the invention in various forms thereof.