US20100317061A1

US20100317061A1 - NOVEL DNA CLONING METHOD RELYING ON THE E.COLI recE/recT RECOMBINATION SYSTEM

Info

Publication number: US20100317061A1
Application number: US12/757,129
Authority: US
Inventors: Francis Stewart; Youming Zhang; Frank Buchholz
Original assignee: Europaisches Laboratorium fuer Molekularbiologie EMBL
Current assignee: Europaisches Laboratorium fuer Molekularbiologie EMBL
Priority date: 1997-12-05
Filing date: 2010-04-09
Publication date: 2010-12-16
Also published as: US20040203057A1; US20030036198A1; JP4139561B2; US6787316B2; EP1291420A1; EP1034260A2; AU752105B2; EP1291420B1; ATE242320T1; US20120208277A1; DE69815384D1; CA2312474A1; WO1999029837A3; CA2759882A1; EP1034260B1; ES2201567T3; DE69815384T2; US7736851B2; PT1034260E; WO1999029837B1

Abstract

The invention relates to methods for cloning DNA molecules using recE/recT-mediated homologous recombination mechanism between at least two DNA molecules where one DNA molecule is a circular or linear DNA molecule and the second DNA molecule is a circular DNA molecule, and the second DNA molecule contains two regions with sequence homology to the first DNA molecule. Competent cells and vectors are also described.

Description

The invention refers to a novel method for cloning DNA molecules using a homologous recombination mechanism between at least two DNA molecules. Further, novel reagent kits suitable for DNA cloning are provided.
Current methods for cloning foreign DNA in bacterial cells usually comprise the steps of providing a suitable bacterial vector, cleaving said vector with a restriction enzyme and in vitro-inserting a foreign DNA fragment in said vector. The resulting recombinant vectors are then used to transform bacteria. Although such cloning methods have been used successfully for about 20 years they suffer from several drawbacks. These drawbacks are, in particular, that the in vitro steps required for inserting foreign DNA in a vector are often very complicated and time-consuming, if no suitable restriction sites are available on the foreign DNA or the vector.
Furthermore, current methods usually rely on the presence of suitable restriction enzyme cleavage sites in the vector into which the foreign DNA fragment is placed. This imposes two limitations on the final cloning product. First, the foreign DNA fragment can usually only be inserted into the vector at the position of such a restriction site or sites. Thus, the cloning product is limited by the disposition of suitable restriction sites and cloning into regions of the vector where there is no suitable restriction site, is difficult and often imprecise. Second, since restriction sites are typically 4 to 8 base pairs in length, they occur a multiple number of times as the size of the DNA molecules being used increases. This represents a practical limitation to the size of the DNA molecules that can be manipulated by most current cloning techniques. In particular, the larger sizes of DNA cloned into vectors such as cosmids, BACs, PACs and P1s are such that it is usually impractical to manipulate them directly by restriction enzyme based techniques. Therefore, there is a need for providing a new cloning method, from which the drawbacks of the prior art have at least partly been eliminated.
According to the present invention it was found that an efficient homologous recombination mechanism between two DNA molecules occurs at usable frequencies in a bacterial host cell which is capable of expressing the products of the recE and recT genes or functionally related genes such as the redα and redβ genes, or the phage P22 recombination system (Kolodner et al., Mol. Microbiol. 11 (1994) 23-30; Fenton, A. C. and Poteete, A. R., Virology 134 (1984) 148-160; Poteete, A. R. and Fenton, A. C., Virology 134 (1984) 161-167). This novel method of cloning DNA fragments is termed “ET cloning”.
The identification and characterization of the E. coli RecE and RecT proteins is described Gillen et al. (J. Bacteriol. 145 (1981), 521-532) and Hall et al. (J. Bacteriol. 175 (1993), 277-287). Hall and Kolodner (Proc. Natl. Acad. Sci. USA 91 (1994), 3205-3209) disclose in vitro homologous pairing and strand exchange of linear double-stranded DNA and homologous circular single-stranded DNA promoted by the RecT protein. Any references to the use of this method for the cloning of DNA molecules in cells cannot be found therein.
The recET pathway of genetic recombination in E. coli is known (Hall and Kolodner (1994), supra; Gillen et al. (1981), supra). This pathway requires the expression of two genes, recE and recT. The DNA sequence of these genes has been published (Hall et al., supra). The RecE protein is similar to bacteriophage proteins, such as λ exo or λ Redα (Gillen et al., J. Mol. Bio 1.113 (1977), 27-41; Little, J. Biol. Chem. 242 (1967), 679-686; Radding and Carter, J. Biol. Chem. 246 (1971), 2513-2518; Joseph and Kolodner, J. Biol. Chem. 258 (1983), 10418-10424). The RecT protein is similar to bacteriophage proteins, such as λ β-protein or λ Redβ (Hall at al. (1993), supra; Muniyappa and Radding, J. Biol. Chem. 261 (1986), 7472-7478; Kmiec and Hollomon, J. Biol. Chem. 256 (1981), 12636-12639). The content of the above-cited documents is incorporated herein by reference.
Oliner et al. (Nucl. Acids Res. 21 (1993), 5192-5197) describe in vivo cloning of PCR products in E. coli by intermolecular homologous recombination between a linear PCR product and a linearized plasmid vector. Other previous attempts to develop new cloning methods based on homologous recombination in prokaryotes, too, relied on the use of restriction enzymes to linearise the vector (Bubeck et al., Nucleic Acids Res. 21 (1993), 3601-3602; Oliner et al., Nucleic Acids Res. 21 (1993), 5192-5197; Degryse, Gene 170 (1996), 45-50) or on the host-specific recA-dependent recombination system (Hamilton et al., J. Bacteriol. 171 (1989), 4617-4622; Yang et al., Nature Biotech. 15 (1997), 859-865; Dabert and Smith, Genetics 145 (1997), 877-889). These methods are of very limited applicability and are hardly used in practice.
The novel method of cloning DNA according to the present invention does not require in vitro treatments with restriction enzymes or DNA ligases and is therefore fundamentally distinct from the standard methodologies of DNA cloning. The method relies on a pathway of homologous recombination in E. coli involving the recE and recT gene products, or the redα and redβ gene products, or functionally equivalent gene products. The method covalently combines one preferably linear and preferably extrachromosomal DNA fragment, the DNA fragment to be cloned, with one second preferably circular DNA vector molecule, either an episome or the endogenous host chromosome or chromosomes. It is therefore distinct from previous descriptions of cloning in E. coli by homologous recombination which either rely on the use of two linear DNA fragments or different recombination pathways.
The present invention provides a flexible way to use homologous recombination to engineer large DNA molecules including an intact >76 kb plasmid and the E. coli chromosome. Thus, there is practically no limitation of target choice either according to size or site. Therefore, any recipient DNA in a host cell, from high copy plasmid to the genome, is amenable to precise alteration. In addition to engineering large DNA molecules, the invention outlines new, restriction enzyme-independent approaches to DNA design. For example, deletions between any two chosen base pairs in a target episome can be made by choice of oligonucleotide homology arms. Similarly, chosen DNA sequences can be inserted at a chosen base pair to create, for example, altered protein reading frames. Concerted combinations of insertions and deletions, as well as point mutations, are also possible. The application of these strategies is particularly relevant to complex or difficult DNA constructions, for example, those intended for homologous recombinations in eukaryotic cells, e.g. mouse embryonic stem cells. Further, the present invention provides a simple way to position site specific recombination target sites exactly where desired. This will simplify applications of site specific recombination in other living systems, such as plants and mice.
A subject matter of the present invention is a method for cloning DNA molecules in cells comprising the steps:

- a) providing a host cell capable of performing homologous recombination,
- b) contacting in said host cell a first DNA molecule which is capable of being replicated in said host cell with a second DNA molecule comprising at least two regions of sequence homology to regions on the first DNA molecule, under conditions which favour homologous recombination between said first and second DNA molecules and
- c) selecting a host cell in which homologous recombination between said first and second DNA molecules has occurred.

In the method of the present invention the homologous recombination preferably occurs via the recET mechanism, i.e. the homologous recombination is mediated by the gene products of the recE and the recT genes which are preferably selected from the E. coli genes recE and recT or functionally related genes such as the phage λ redα and redβ genes.
The host cell suitable for the method of the present invention preferably is a bacterial cell, e.g. a gram-negative bacterial cell. More preferably, the host cell is an enterobacterial cell, such as Salmonella, Klebsiella or Escherichia. Most preferably the host cell is an Escherichia coli cell. It should be noted, however, that the cloning method of the present invention is also suitable for eukaryotic cells, such as fungi, plant or animal cells.
Preferably, the host cell used for homologous recombination and propagation of the cloned. DNA can be any cell, e.g. a bacterial strain in which the products of the recE and recT, or redα and redβ, genes are expressed. The host cell may comprise the recE and recT genes located on the host cell chromosome or on non-chromosomal DNA, preferably on a vector, e.g. a plasmid. In a preferred case, the RecE and RecT, or Redα and Redβ, gene products are expressed from two different regulatable promoters, such as the arabinose-inducible BAD promoter or the lac promoter or from non-regulatable promoters. Alternatively, the recE and recT, or redα and redβ, genes are expressed on a polycistronic mRNA from a single regulatable or non-regulatable promoter. Preferably the expression is controlled by regulatable promoters.
Especially preferred is also an embodiment, wherein the recE or redα gene is expressed by a regulatable promoter. Thus, the recombinogenic potential of the system is only elicited when required and, at other times, possible undesired recombination reactions are limited. The recT or redβ gene, on the other hand, is preferably overexpressed with respect to recE or redα. This may be accomplished by using a strong constitutive promoter, e.g. the EM7 promoter and/or by using a higher copy number of recT, or redβ, versus recE, or redα, genes.
For the purpose of the present invention any recE and recT genes are suitable insofar as they allow a homologous recombination of first and second DNA molecules with sufficient efficiency to give rise to recombination products in more than 1 in 10⁹cells transfected with DNA. The recE and recT genes may be derived from any bacterial strain or from bacteriophages or may be mutants and variants thereof. Preferred are recE and recT genes which are derived from E. coli or from E. coli bacteriophages, such as the redα and redβ genes from lambdoid phages, e.g. bacteriophage λ.
More preferably, the recE or redα gene is selected from a nucleic acid molecule comprising
(a) the nucleic acid sequence from position 1320 (ATG) to 2159 (GAC) as depicted in FIG. 7B,
(b) the nucleic acid sequence from position 1320 (ATG) to 1998(CGA) as depicted in FIG. 14B,
(c) a nucleic acid encoding the same polypeptide within the degeneracy of the genetic code and/or
(d) a nucleic acid sequence which hybridizes under stringent conditions with the nucleic acid sequence from (a), (b) and/or (c).
More preferably, the recT or redβ gene is selected from a nucleic acid molecule comprising
(a) the nucleic acid sequence from position 2155 (ATG) to 2961 (GAA) as depicted in FIG. 7B,
(b) the nucleic acid sequence from position 2086 (ATG) to 2868 (GCA) as depicted in FIG. 14B,
(c) a nucleic acid encoding the same polypeptide within the degeneracy of the genetic code and/or
(d) a nucleic acid sequence which hybridizes under stringent conditions with the nucleic acid sequences from (a), (b) and/or (c).
It should be noted that the present invention also encompasses mutants and variants of the given sequences, e.g. naturally occurring mutants and variants or mutants and variants obtained by genetic engineering. Further it should be noted that the recE gene depicted in FIG. 7B is an already truncated gene encoding amino acids 588-866 of the native protein. Mutants and variants preferably have a nucleotide sequence identity of at least 60%, preferably of at least 70% and more preferably of at least 80% of the recE and recT sequences depicted in FIGS. 7B and 13B, and of the redα and redβ sequences depicted in FIG. 14B.
According to the present invention hybridization under stringent conditions preferably is defined according to Sambrook et al. (1989), infra, and comprises a detectable hybridization signal after washing for 30 min in 0.1×SSC, 0.5% SDS at 55° C., preferably at 62° C. and more preferably at 68° C.
In a preferred case the recE and recT genes are derived from the corresponding endogenous genes present in the E. coli K12 strain and its derivatives or from bacteriophages. In particular, strains that carry the sbcA mutation are suitable. Examples of such strains are JC8679 and JC 9604 (Gillen et al. (1981), supra). Alternatively, the corresponding genes may also be obtained from other coliphages such as lambdoid phages or phage P22.
The genotype of JC 8679 and JC 9604 is Sex (Hfr, F+, F−, or F′): F-.JC 8679 comprises the mutations: recBC 21, recC 22, sbcA 23, thr-1, ara-14, leu B 6, DE (gpt-proA) 62, lacY1, tsx-33, gluV44 (AS), galK2 (Oc), LAM-, his-60, relA 1, rps L31 (strR), xyl A5, mtl-1, argE3 (Oc) and thi-1. JC 9604 comprises the same mutations and further the mutation recA 56.
Further, it should be noted that the recE and recT, or redα and redβ, genes can be isolated from a first donor source, e.g. a donor bacterial cell and transformed into a second receptor source, e.g. a receptor bacterial or eukaryotic cell in which they are expressed by recombinant DNA means.
In one embodiment of the invention, the host cell used is a bacterial strain having an sbcA mutation, e.g. one of E. coli strains JC 8679 and JC 9604 mentioned above. However, the method of the invention is not limited to host cells having an sbcA mutation or analogous cells. Surprisingly, it has been found that the cloning method of the invention also works in cells without sbcA mutation, whether recBC+ or recBC−, e.g. also in prokaryotic recBC+ host cells, e.g. in E. coli recBC+ cells. In that case preferably those host cells are used in which the product of a recBC type exonuclease inhibitor gene is expressed. Preferably, the exonuclease inhibitor is capable of inhibiting the host recBC system or an equivalent thereof. A suitable example of such exonuclease inhibitor gene is the λ redγ gene (Murphy, J. Bacteriol. 173 (1991), 5808-5821) and functional equivalents thereof, respectively, which, for example, can be obtained from other coliphages such as from phage P22 (Murphy, J. Biol. Chem. 269 (1994), 22507-22516).
More preferably, the exonuclease inhibitor gene is selected from a nucleic acid molecule comprising
(a) the nucleic acid sequence from position 3588 (ATG) to 4002 (GTA) as depicted in FIG. 14A,
(b) a nucleic acid encoding the same polypeptide within the degeneracy of the genetic code and/or
(c) a nucleic acid sequence which hybridizes under stringent conditions (as defined above) with the nucleic acid sequence from (a) and/or (b).
Surprisingly, it has been found that the expression of an exonuclease inhibitor gene in both recBC+ and recBC− strains leads to significant improvement of cloning efficiency.
The cloning method according to the present invention employs a homologous recombination between a first DNA molecule and a second DNA molecule. The first DNA molecule can be any DNA molecule that carries an origin of replication which is operative in the host cell, e.g. an E. coli replication origin. Further, the first DNA molecule is present in a form which is capable of being replicated in the host cell. The first DNA molecule, i.e. the vector, can be any extrachromosomal DNA molecule containing an origin of replication which is operative in said host cell, e.g. a plasmid including single, low, medium or high copy plasmids or other extrachromosomal circular DNA molecules based on cosmid, P1, BAC or PAC vector technology. Examples of such vectors are described, for example, by Sambrook et al. (Molecular Cloning, Laboratory Manual, 2nd Edition (1989), Cold Spring Harbor Laboratory Press) and loannou et al. (Nature Genet. 6 (1994), 84-89) or references cited therein. The first DNA molecule can also be a host cell chromosome, particularly the E. coli chromosome. Preferably, the first DNA molecule is a double-stranded DNA molecule.
The second DNA molecule is preferably a linear DNA molecule and comprises at least two regions of sequence homology, preferably of sequence identity to regions on the first DNA molecule. These homology or identity regions are preferably at least 15 nucleotides each, more preferably at least 20 nucleotides and, most preferably, at least 30 nucleotides each. Especially good results were obtained when using sequence homology regions having a length of about 40 or more nucleotides, e.g. 60 or more nucleotides. The two sequence homology regions can be located on the linear DNA fragment so that one is at one end and the other is at the other end, however they may also be located internally. Preferably, also the second DNA molecule is a double-stranded DNA molecule.
The two sequence homology regions are chosen according to the experimental design. There are no limitations on which regions of the first DNA molecule can be chosen for the two sequence homology regions located on the second DNA molecule, except that the homologous recombination event cannot delete the origin of replication of the first DNA molecule. The sequence homology regions can be interrupted by non-identical sequence regions as long as sufficient sequence homology is retained for the homologous recombination reaction. By using sequence homology arms having non-identical sequence regions compared to the target site mutations such as substitutions, e.g. point mutations, insertions and/or deletions may be introduced into the target site by ET cloning.
The second foreign DNA molecule which is to be cloned in the bacterial cell may be derived from any source. For example, the second DNA molecule may be synthesized by a nucleic acid amplification reaction such as a PCR where both of the DNA oligonucleotides used to prime the amplification contain in addition to sequences at the 3′-ends that serve as a primer for the amplification, one or the other of the two homology regions. Using oligonucleotides of this design, the DNA product of the amplification can be any DNA sequence suitable for amplification and will additionally have a sequence homology region at each end.
A specific example of the generation of the second DNA molecule is the amplification of a gene that serves to convey a phenotypic difference to the bacterial host cells, in particular, antibiotic resistance. A simple variation of this procedure involves the use of oligonucleotides that include other sequences in addition to the PCR primer sequence and the sequence homology region. A further simple variation is the use of more than two amplification primers to generate the amplification product. A further simple variation is the use of more than one amplification reaction to generate the amplification product. A further variation is the use of DNA fragments obtained by methods other than PCR, for example, by endonuclease or restriction enzyme cleavage to linearize fragments from any source of DNA.
It should be noted that the second DNA molecule is not necessarily a single species of DNA molecule. It is of course possible to use a heterogenous population of second DNA molecules, e.g. to generate a DNA library, such as a genomic or cDNA library.
The method of the present invention may comprise the contacting of the first and second DNA molecules in vivo. In one embodiment of the present invention the second DNA fragment is transformed into a bacterial strain that already harbors the first vector DNA molecule. In a different embodiment, the second DNA molecule and the first DNA molecule are mixed together in vitro before co-transformation in the bacterial host cell. These two embodiments of the present invention are schematically depicted in FIG. 1. The method of transformation can be any method known in the art (e.g. Sambrook et al. supra). The preferred method of transformation or co-transformation, however, is electroporation.
After contacting the first and second DNA molecules under conditions which favour homologous recombination between first and second DNA molecules via the ET cloning mechanism a host cell is selected, in which homologous recombination between said first and second DNA molecules has occurred. This selection procedure can be carried out by several different methods. In the following three preferred selection methods are depicted in FIG. 2 and described in detail below.
In a first selection method a second DNA fragment is employed which carries a gene for a marker placed between the two regions of sequence homology wherein homologous recombination is detectable by expression of the marker gene. The marker gene may be a gene for a phenotypic marker which is not expressed in the host or from the first DNA molecule. Upon recombination by ET cloning, the change in phenotype of the host strain conveyed by the stable acquisition of the second DNA fragment identifies the ET cloning product.
In a preferred case, the phenotypic marker is a gene that conveys resistance to an antibiotic, in particular, genes that convey resistance to kanamycin, ampillicin, chloramphenicol, tetracyclin or any other substance that shows bacteriocidal or bacteriostatic effects on the bacterial strain employed.
A simple variation is the use of a gene that complements a deficiency present within the bacterial host strain employed. For example, the host strain may be mutated so that it is incapable of growth without a metabolic supplement. In the absence of this supplement, a gene on the second DNA fragment can complement the mutational defect thus permitting growth. Only those cells which contain the episome carrying the intended DNA rearrangement caused by the ET cloning step will grow.
In another example, the host strain carries a phenotypic marker gene which is mutated so that one of its codons is a stop codon that truncates the open reading frame. Expression of the full length protein from this phenotypic marker gene requires the introduction of a suppressor tRNA gene which, once expressed, recognizes the stop codon and permits translation of the full open reading frame. The suppressor tRNA gene is introduced by the ET cloning step and successful recombinants identified by selection for, or identification of, the expression of the phenotypic marker gene. In these cases, only those cells which contain the intended DNA rearrangement caused by the ET cloning step will grow.
A further simple variation is the use of a reporter gene that conveys a readily detectable change in colony colour or morphology. In a preferred case, the green fluorescence protein (GFP) can be used and colonies carrying the ET cloning product identified by the fluorescence emissions of, GFP. In another preferred case, the lacZ gene can be used and colonies carrying the ET cloning product identified by a blue colony colour when X-gal is added to the culture medium.
In a second selection method the insertion of the second DNA fragment into the first DNA molecule by ET cloning alters the expression of a marker present on the first DNA molecule. In this embodiment the first DNA molecule contains at least one marker gene between the two regions of sequence homology and homologous recombination may be detected by an altered expression, e.g. lack of expression of the marker gene.
In a preferred application, the marker present on the first DNA molecule is a counter-selectable gene product, such as the sacB, ccdB or tetracycline-resistance genes. In these cases, bacterial cells that carry the first DNA molecule unmodified by the ET cloning step after transformation with the second DNA fragment, or co-transformation with the second DNA fragment and the first DNA molecule, are plated onto a medium so the expression of the counter-selectable marker conveys a toxic or bacteriostatic effect on the host. Only those bacterial cells which contain the first DNA molecule carrying the intended DNA rearrangement caused by the ET cloning step will grow.
In another preferred application, the first DNA molecule carries a reporter gene that conveys a readily detectable change in colony colour or morphology. In a preferred case, the green fluorescence protein (GFP) can be present on the first DNA molecule and colonies carrying the first DNA molecule with or without the ET cloning product can be distinguished by differences in the fluorescence emissions of GFP. In another preferred case, the lacZ gene can be present on the first DNA molecule and colonies carrying the first DNA molecule with or without the ET cloning product identified by a blue or white colony colour when X-gal is added to the culture medium.
In a third selection method the integration of the second DNA fragment into the first DNA molecule by ET cloning removes a target site for a site specific recombinase, termed here an RT (for recombinase target) present on the first DNA molecule between the two regions of sequence homology. A homologous recombination event may be detected by removal of the target site.
In the absence of the ET cloning product, the RT is available for use by the corresponding site specific recombinase. The difference between the presence or not of this RT is the basis for selection of the ET cloning product. In the presence of this RT and the corresponding site specific recombinase, the site specific recombinase mediates recombination at this RT and changes the phenotype of the host so that it is either not able to grow or presents a readily observable phenotype. In the absence of this RT, the corresponding site specific recombinase is not able to mediate recombination.
In a preferred case, the first DNA molecule to which the second DNA fragment is directed, contains two RTs, one of which is adjacent to, but not part of, an antibiotic resistance gene. The second DNA fragment is directed, by design, to remove this RT. Upon exposure to the corresponding site specific recombinase, those first DNA molecules that do not carry the ET cloning product will be subject to a site specific recombination reaction between the RTs that remove the antibiotic resistance gene and therefore the first DNA molecule fails to convey resistance to the corresponding antibiotic. Only those first DNA molecules that contain the ET cloning product, or have failed to be site specifically recombined for some other reason, will convey resistance to the antibiotic.
In another preferred case, the RT to be removed by ET cloning of the second DNA fragment is adjacent to a gene that complements a deficiency present within the host strain employed. In another preferred case, the RT to be removed by ET cloning of the second DNA fragment is adjacent to a reporter gene that conveys a readily detectable change in colony colour or morphology.
In another preferred case, the RT to be removed by ET cloning of the second DNA fragment is anywhere on a first episomal DNA molecule and the episome carries an origin of replication incompatible with survival of the bacterial host cell if it is integrated into the host genome. In this case the host genome carries a second RT, which may or may not be a mutated RT so that the corresponding site specific recombinase can integrate the episome, via its RT, into the RT sited in the host genome. Other preferred. RTs include RTs for site specific recombinases of the resolvase/transposase class. RTs include those described from existing examples of site specific recombination as well as natural or mutated variations thereof.
The preferred site specific recombinases include Cre, FLP, Kw or any site specific recombinase of the integrase class. Other preferred site specific recombinases include site specific recombinases of the resolvase/transposase class.
There are no limitations on the method of expression of the site specific recombinase in the host cell. In a preferred method, the expression of the site specific recombinase is regulated so that expression can be induced and quenched according to the optimisation of the ET cloning efficiency. In this case, the site specific recombinase gene can be either integrated into the host genome or carried on an episome. In another preferred case, the site specific recombinase is expressed from an episome that carries a conditional origin of replication so that it can be eliminated from the host cell.
In another preferred case, at least two of the above three selection methods are combined. A particularly preferred case involves a two-step use of the first selection method above, followed by use of the second selection method. This combined use requires, most simply, that the DNA fragment to be cloned includes a gene, or genes that permits the identification, in the first step, of correct ET cloning products by the acquisition of a phenotypic change. In a second step, expression of the gene or genes introduced in the first step is altered so that a second round of ET cloning products can be identified. In a preferred example, the gene employed is the tetracycline resistance gene and the first step ET cloning products are identified by the acquisition of tetracycline resistance. In the second step, loss of expression of the tetracycline gene is identified by loss of sensitivity to nickel chloride, fusaric acid or any other agent that is toxic to the host cell when the tetracycline gene is expressed. This two-step procedure permits the identification of ET cloning products by first the integration of a gene that conveys a phenotypic change on the host, and second by the loss of a related phenotypic change, most simply by removal of some of the DNA sequences integrated in the first step. Thereby the genes used to identify ET cloning products can be inserted and then removed to leave ET cloning products that are free of these genes.
In a further embodiment of the present invention the ET cloning may also be used for a recombination method comprising the steps of
a) providing a source of RecE and RecT, or Redα and Redβ, proteins,
b) contacting a first DNA molecule which is capable of being replicated in a suitable host cell with a second DNA molecule comprising at least two regions of sequence homology to regions on the first DNA molecule, under conditions which favour homologous recombination between said first and second DNA molecules and
c) selecting DNA molecules in which a homologous recombination between said first and second DNA molecules has occurred.
The source of RecE and RecT, or Redα and Redβ, proteins may be either purified or partially purified RecE and RecT, or Redα and Redβ, proteins or cell extracts comprising RecE and RecT, or Redα and Redβ, proteins.
The homologous recombination event in this embodiment may occur in vitro, e.g. when providing a cell extract containing further components required for homologous recombination. The homologous recombination event, however, may also occur in vivo, e.g. by introducing RecE and RecT, or Redα and Redβ, proteins or the extract in a host cell (which may be recET positive or not, or redαβ positive or not) and contacting the DNA molecules in the host cell. When the recombination occurs in vitro the selection of DNA molecules may be accomplished by transforming the recombination mixture in a suitable host cell and selecting for positive clones as described above. When the recombination occurs in vivo the selection methods as described above may directly be applied.
A further subject matter of the invention is the use of cells, preferably bacterial cells, most preferably, E. coli cells capable of expressing the recE and recT, or redα and redβ, genes as a host cell for a cloning method involving homologous recombination.
Still a further subject matter of the invention is a vector system capable of expressing recE and recT, or redα and redβ, genes in a host cell and its use for a cloning method involving homologous recombination. Preferably, the vector system is also capable of expressing an exonuclease inhibitor gene as defined above, e.g. the λ redγ gene. The vector system may comprise at least one vector. The recE and recT, or redα and redβ, genes are preferably located on a single vector and more preferably under control of a regulatable promoter which may be the same for both genes or a single promoter for each gene. Especially preferred is a vector system which is capable of overexpressing the recT, or redβ, gene versus the recE, or redα, gene.
Still a further subject matter of the invention is the use of a source of RecE and RecT, or Redα and Redβ, proteins for a cloning method involving homologous recombination.
A still further subject matter of the invention is a reagent kit for cloning comprising

- (a) a host cell, preferably a bacterial host cell,
- (b) means of expressing recE and recT, or redα and redβ, genes in said host cell, e.g. comprising a vector system, and
- (c) a recipient cloning vehicle, e.g. a vector, capable of being replicated in said cell.

On the one hand, the recipient cloning vehicle which corresponds to the first DNA molecule of the process of the invention can already be present in the bacterial cell. On the other hand, it can be present separated from the bacterial cell.
In a further embodiment the reagent kit comprises
(a) a source for RecE and RecT, or Redα and Red % proteins and
(b) a recipient cloning vehicle capable of being propagated in a host cell and
(c) optionally a host cell suitable for propagating said recipient cloning vehicle.
The reagent kit furthermore contains, preferably, means for expressing a site specific recombinase in said host cell, in particular, when the recipient ET cloning product contains at least one site specific recombinase target site. Moreover, the reagent kit can also contain DNA molecules suitable for use as a source of linear DNA fragments used for ET cloning, preferably by serving as templates for PCR generation of the linear fragment, also as specifically designed DNA vectors from which the linear DNA fragment is released by restriction enzyme cleavage, or as prepared linear fragments included in the kit for use as positive controls or other tasks. Moreover, the reagent kit can also contain nucleic acid amplification primers comprising a region of homology to said vector. Preferably, this region of homology is located at the 5′-end of the nucleic acid amplification primer.
The invention is further illustrated by the following Sequence listings, Figures and Examples.

SEQ ID NO. 1: shows the nucleic acid sequence of the plasmid pBAD24-rec ET (FIG. 7).
SEQ ID NOs 2/3: show the nucleic acid and amino acid sequences of the truncated recE gene (t-recE) present on pBAD24-recET at positions 1320-2162.
SEQ ID NOs 4/5: show the nucleic acid and amino acid sequences of the recT gene present on pBAD24-recET at position 2155-2972.
SEQ ID NOs 6/7: show the nucleic acid and amino acid sequences of the araC gene present on the complementary stand to the one shown of pBAD24-recET at positions 974-996.
SEQ ID NOs 8/9: show the nucleic acid an amino acid sequences of the bla gene present on pBAD24-recET at positions 3493-4353.
SEQ ID NO 10: shows the nucleic acid sequence of the plasmid pBAD-ETγ (FIG. 13).
SEQ ID No 11: shows the nucleic acid sequence of the plasmid pBAD-αβγ (FIG. 14) as well as the coding regions for the genes redα (1320-200), redβ (2086-2871) and redγ (3403-3819).
SEQ ID NOs 12-14: show the amino acid sequences of the Redα, Redβ and Redγ proteins, respectively. The redγ sequence is present on each of pBAD-ETγ (FIG. 13) and pBAD-αβγ (FIG. 14).

FIG. 1

A preferred method for ET cloning is shown by diagram. The linear DNA fragment to be cloned is synthesized by PCR using oligonucleotide primers that contain a left homology arm chosen to match sequences in the recipient episome and a sequence for priming in the PCR reaction, and a right homology arm chosen to match another sequence in the recipient episome and a sequence for priming in the PCR reaction. The product of the PCR reaction, here a selectable marker gene (sm1), is consequently flanked by the left and right homology arms and can be mixed together in vitro with the episome before co-transformation, or transformed into a host cell harboring the target episome. The host cell contains the products of the recE and recT genes. ET cloning products are identified by the combination of two selectable markers, sm 1 and sm2 on the recipient episome.

FIG. 2

Three ways to identify ET cloning products are depicted. The first, (on the left of the figure), shows the acquisition, by ET cloning, of a gene that conveys a phenotypic difference to the host, here a selectable marker gene (sm). The second (in the centre of the figure) shows the loss, by ET cloning, of a gene that conveys a phenotypic difference to the host, here a counter selectable marker gene (counter-sm). The third shows the loss of a target site (RT, shown as triangles on the circular episome) for a site specific recombinase (SSR), by ET cloning. In this case, the correct ET cloning product deletes one of the target sites required by the SSR to delete a selectable marker gene (sm). The failure of the SSR to delete the sm gene identifies the correct ET cloning product.

FIG. 3

A simple example of ET cloning is presented.
(a) Top panel—PCR products (left lane) synthesized from oligonucleotides designed as described in FIG. 1 to amplify by PCR a kanamycin resistance gene and to be flanked by homology arms present in the recipient vector, were mixed in vitro with the recipient vector (2nd lane) and cotransformed into a recET+E. coli host. The recipient vector carried an ampillicin resistance gene. (b) Transformation of the sbcA E. coli strain JC9604 with either the PCR product alone (0.2 μg) or the vector alone (0.3 μg) did not convey resistance to double selection with ampicillin and kanamycin (amp+kan), however cotransformation of both the PCR product and the vector produced double resistant colonies. More than 95% of these colonies contained the correct ET cloning product where the kanamycin gene had precisely integrated into the recipient vector according to the choice of homology arms. The two lanes on the right of (a) show Pvu II restriction enzyme digestion of the recipient vector before and after ET cloning. (c) As for b, except that six PCR products (0.2 μg each) were cotransformed with pSVpaZ11 (0.3 μg each) into JC9604 and plated onto Amp+Kan plates or Amp plates. Results are plotted as Amp+Kan-resistant colonies, representing recombination products, divided by Amp-resistant colonies, representing the plasmid transformation efficiency of the competent cell preparation, ×10⁶. The PCR products were equivalent to the a-b PCR product except that homology arm lengths were varied. Results are from five experiments that used the same batches of competent cells and DNAs. Error bars represent standard deviation. (d) Eight products flanked by 50 by homology arms were cotransformed with pSVpaZ11 into JC9604. All eight PCR products contained the same left homology arm and amplified neo gene. The right homology arms were chosen from the pSVpaZ11 sequence to be adjacent to (O), or at increasing distances (7-3100 bp), from the left. Results are from four experiments.

FIG. 4

ET cloning in an approximately 100 kb P1 vector to exchange the selectable marker.
A P1 clone which uses a kanamycin resistance gene as selectable marker and which contains at least 70 kb of the mouse Hox a gene cluster was used. Before ET cloning, this episome conveys kanamycin resistance (top panel, upper left) to its host E. coli which are ampillicin sensitive (top panel, upper right). A linear DNA fragment designed to replace the kanamycin resistance gene with an ampillicin resistance gene was made by PCR as outlined in FIG. 1 and transformed into E. coli host cells in which the recipient Hox a/P1 vector was resident. ET cloning resulted in the deletion of the kanamycin resistance gene, and restoration of kanamycin sensitivity (top panel, lower left) and the acquisition of ampillicin resistance (top panel, lower right). Precise DNA recombination was verified by restriction digestion and Southern blotting analyses of isolated DNA before and after ET cloning (lower panel).

FIG. 5

ET cloning to remove a counter selectable marker
A PCR fragment (upper panel, left, third lane) made as outlined in FIGS. 1 and 2 to contain the kanamycin resistance gene was directed by its chosen homology arms to delete the counter selectable ccdB gene present in the vector, pZero-2.1. The PCR product and the pZero vector were mixed in vitro (upper panel, left, 1st lane) before cotransformation into a recE/recT+E. coli host. Transformation of pZero-2.1 alone and plating onto kanamycin selection medium resulted in little colony growth (lower panel, left). Cotransformation of pZero-2.1 and the PCR product presented ET cloning products (lower panel, right) which showed the intended molecular event as visualized by Pvu II digestion (upper panel, right).

FIG. 6

ET cloning mediated by inducible expression of recE and recT from an episome.
RecE/RecT mediate homologous recombination between linear and circular DNA molecules. (a) The plasmid pBAD24-recET was transformed into E. coli JC5547, and then batches of competent cells were prepared after induction of RecE/RecT expression by addition of L-arabinose for the times indicated before harvesting. A PCR product, made using oligonucleotides e and f to contain the chloramphenicol resistance gene (cm) of pMAK705 and 50 bp homology arms chosen to flank the ampicililin resistance gene (bla) of pBAD24-recET, was then transformed and recombinants identified on chloramphenicol plates. (b) Arabinose was added to cultures of pBAD24-recET transformed JC5547 for different times immediately before harvesting for competent cell preparation. Total protein expression was analyzed by SDS-PAGE and Coomassie blue staining. (c) The number of chloramphenicol resistant colonies per μg of PCR product was normalized against a control for transformation efficiency, determined by including 5 μg pZero2.1, conveying kanamycin resistance, in the transformation and plating an aliquot onto Kan plates.

FIG. 7A

The plasmid pBAD24-recET is shown by diagram. The plasmid contains the genes recE (in a truncated form) and recT under control of the inducible BAD promoter (P_BAD). The plasmid further contains an ampillicin resistance gene (Amp′) and an araC gene.

FIG. 7B

The nucleic acid sequence and the protein coding portions of pBAD24-recET are depicted.

FIG. 8

Manipulation of a large E. coli episome by multiple recombination steps. a Scheme of the recombination reactions. A P1 clone of the Mouse Hoxa complex, resident in JC9604, was modified by recombination with PCR products that contained the neo gene and two Flp recombination targets (FRTs). The two PCR products were identical except that one was flanked by g and h homology arms (insertion), and the other was flanked by i and h homology arms (deletion). In a second step, the neo gene was removed by Flp recombination between the FRTs by transient transformation of a Flp expression plasmid based on the pSC101 temperature-sensitive origin (ts ori). b Upper panel; ethidium bromide stained agarose gel showing EcoR1 digestions of P1 DNA preparations from three independent colonies for each step. Middle panel; a Southern blot of the upper panel hybridized with a neo gene probe. Lower panel; a Southern blot of the upper panel hybridized with a Hoxa3 probe to visualize the site of recombination. Lanes 1, the original Hoxa3 P1 clone grown in E. coli strain NS3145. Lanes 2, replacement of the Tn903 kanamycin resistance gene resident in the P1 vector with an ampicillin resistance gene increased the 8.1 kb band (lanes 1), to 9.0 kb. Lanes 3, insertion of the Tn5-neo gene with g-h homology arms upstream of Hoxa3, increased the 6.7 kb band (lanes 1, 2) to 9.0 kb. Lanes 4, Flp recombinase deleted the g-h neo gene reducing the 9.0 kb band (lanes 3) back to 6.7 kb. Lanes 5, deletion of 6 kb of Hoxa3-4 intergenic DNA by replacement with the i-h neo gene, decreased the 6.7 kb band (lanes 2) to 4.5 kb. Lanes 6, Flp recombinase deleted the i-h neo gene reducing the 4.5 kb band to 2.3 kb.

FIG. 9

Manipulation of the E. coli chromosome. A Scheme of the recombination reactions. The endogenous lacZ gene of JC9604 at 7.8′ of the E. coli chromosome, shown in expanded form with relevant Ava I sites and coordinates, was targeted by a PCR fragment that contained the neo gene flanked by homology arms j and k, and loxP sites, as depicted. Integration of the neo gene removed most of the lacZ gene including an Ava I site to alter the 1443 and 3027 by bands into a 3277 by band. In a second step, the neo gene was removed by Cre recombination between the IoxPs by transient transformation of a Cre expression plasmid based on the pSC101temperature-sensitive origin (ts ori). Removal of the neo gene by Cre recombinase reduces the 3277 band to 2111 bp. b β-galactosidase expression evaluated by streaking colonies on X-Gal plates. The top row of three streaks show β-galactosidase expression in the host JC9604 strain (w.t.), the lower three rows (Km) show 24 independent primary colonies, 20 of which display a loss of β-galactosidase expression indicactive of the intended recombination event. c Southern analysis of E. coli chromosomal DNA digested with Ava I using a random primed probe made from the entire lacZ coding region; lanes 1, 2, w.t.; lanes 3-6, four independent white colonies after integration of the j-k neo gene; lanes 7-10; the same four colonies after transient transformation with the Cre expression plasmid.

FIG. 10

Two rounds of ET cloning to introduce a point mutation. a Scheme of the recombination reactions. The lacZ gene of pSVpaX1 was disrupted in JC9604lacZ, a strain made by the experiment of FIG. 9 to ablate endogenous lacZ expression and remove competitive sequences, by a sacB-neo gene cassette, synthesized by PCR to plB279 and flanked by I and m homology arms. The recombinants, termed pSV-sacB-neo, were selected on Amp+Kan plates. The lacZ gene of pSV-sacB-neo was then repaired by a PCR fragment made from the intact lacZ gene using l* and m* homology arms. The m* homology arm included a silent C to G change that created a BamH1 site. The recombinants, termed pSVpaX1′, were identified by counter selection against the sacB gene using 7% sucrose. b β-galactosidase expression from pSVpaX1 was disrupted in pSV-sacB-neo and restored in pSVpaX1′. Expression was analyzed on X-gal plates. Three independent colonies of each pSV-sacB-neo and pSVpaX1′ are shown. c Ethidium bromide stained agarose gels of BamH1 digested DNA prepared from independent colonies taken after counter selection with sucrose. All β-galactosidase expressing colonies (blue) contained the introduced BamH1 restriction site (upper panel). All white colonies displayed large rearrangements and no product carried the diagnostic 1.5 kb BamH1 restriction fragment (lower panel).

FIG. 11

Transferance of ET cloning into a recBC+host to modify a large episome. a Scheme of the plasmid, pBAD-ETγ, which carries the mobile ET system, and the strategy employed to target the Hoxa P1 episome. pBAD-ETγ is based on pBAD24 and includes (i) the truncated recE gene (t-recE) under the arabinose-inducible P_BADpromoter; (ii) the recT gene under the EM7 promoter; and (iii) the redγ gene under the Tn5 promoter. It was transformed into NS3145, a recA E. coli strain which contained the Hoxa P1 episome. After arabinose induction, competent cells were prepared and transformed with a PCR product carrying the chloramphenicol resistance gene (cm) flanked by n and p homology arms. n and p were chosen to recombine with a segment of the P1 vector. b Southern blots of Pvu II digested DNAs hybridized with a probe made from the P1 vector to visualize the recombination target site (upper panel) and a probe made from the chloramphenicol resistance gene (lower panel). Lane 1, DNA prepared from cells harboring the Hoxa P1 episome before ET cloning. Lanes 2-17, DNA prepared from 16 independent chloramphenicol resistant colonies.

FIG. 12

Comparison of ET cloning using the recE/recT genes in pBAD-ETγ with redα/redβ genes in pBAD-αβγ.
The plasmids pBAD-ETγ or pBAD-αβγ, depicted, were transformed into the E. coli recA−, recBC+strain, DK1 and targeted by a chloramphenicol gene as described in FIG. 6 to evaluate ET cloning efficiencies. Arabinose induction of protein expression was for 1 hour.

FIG. 13A

The plasmid pBAD-ETγ is shown by diagram.

FIG. 13B

The nucleic acid sequence and the protein coding portions of pBAD-ETγ are depicted.

FIG. 14A

The plasmid pBAD-αβγ is shown by diagram. This plasmid substantially corresponds to the plasmid shown in FIG. 13 except that the recE and recT genes are substituted by the redα and redβ genes.

FIG. 14B

The nucleic acid sequence and the protein coding portions of pBAD-αβγ are depicted.

1. Methods

1.1. Preparation of Linear Fragments

Standard PCR reaction conditions were used to amplify linear DNA fragments. The sequences of the primers used are depicted in Table 1.

Table 1

The Tn5-neo gene from pJP5603 (Penfold and Pemberton, Gene 118 (1992), 145-146) was amplified by using oligo pairs a/b and c/d. The chloramphenicol (cm) resistant gene from pMAK705 (Hashimoto-Gotoh and Sekiguchi, J. Bacteriol. 131 (1977), 405-412) was amplified by using primer pairs e/f and n/p. The Tn5-neo gene flanked by FRT or loxP sites was amplified from pKaZ or pKaX (http://www.embl-heidelberg.de/ExternalInfo/stewart) using oligo pairs i/h, g/h and j/k. The sacB-neo cassette from plB279 (Blomfield et al., Mol. Microbiol. 5 (1991), 1447-1457) was amplified by using oligo pair l/m. The lacZ gene fragment from pSVpaZ11 (Buchholz et al., Nucleic Acids Res. 24 (1996), 4256-4262) was amplified using oligo pair l*/m*. PCR products were purified using the QIAGEN PCR Purification Kit and eluted with H₂O₂, followed by digestion of any residual template DNA with Dpn I. After digestion, PCR products were extracted once with Phenol:CHCl₃, ethanol precipitated and resuspended in H₂O at approximately 0.5 μg/μl.

1.2 Preparation of Competent Cells and Electroporation

Saturated overnight cultures were diluted 50 fold into LB medium, grown to an OD600 of 0.5, following by chilling on ice for 15 min. Bacterial cells were centrifuged at 7,000 rpm for 10 min at 0° C. The pellet was resuspended in ice-cold 10% glycerol and centrifuged again (7,000 rpm, −5° C., 10 min). This was repeated twice more and the cell pellet was suspended in an equal volume of ice-cold 10% glycerol. Aliquots of 50 μl were frozen in liquid nitrogen and stored at −80° C. Cells were thawed on ice and 1 μl DNA solution (containing, for co-transformation, 0.3 μg plasmid and 0.2 μg PCR products; or, for transformation, 0.2 μg PCR products) was added. Electroporation was performed using ice-cold cuvettes and a Bio-Rad Gene Pulser set to 25 μFD, 2.3 kV with Pulse Controller set at 200 ohms. LB medium (1 ml) was added after electroporation. The cells were incubated at 37° C. for 1 hour with shaking and then spread on antibiotic plates.

1.3 Induction of RecE and RecT Expression

E. coli JC5547 carrying pBAD24-recET was cultured overnight in LB medium plus 0.2% glucose, 100 μg/ml ampicillin. Five parallel LB cultures, one of which (O) included 0.2% glucose, were started by a 1/100 inoculation. The cultures were incubated at 37° C. with shaking for 4 hours and 0.1% L-arabinose was added 3, 2, 1 or ½ hour before harvesting and processing as above. Immediately before harvesting, 100 μl was removed for analysis on a 10% SDS-polyacrylamide gel. E. coli NS3145 carrying Hoxa-P1 and pBAD-ETγ was induced by 0.1% L-arabinose for 90 min before harvesting.

1.4 Transient Transformation of FLP and Cre Expression Plasmids

The FLP and Cre expression plasmids, 705-Cre and 705-FLP (Buchholz et al, Nucleic Acids Res. 24 (1996), 3118-3119), based on the pSC101 temperature sensitive origin, were transformed into rubidium chloride competent bacterial cells. Cells were spread on 25 μg/ml chloramphenicol plates, and grown for 2 days at 30° C., whereupon colonies were picked, replated on L-agar plates without any antibiotics and incubated at 40° C. overnight. Single colonies were analyzed on various antibiotic plates and all showed the expected loss of chloramphenicol and kanamycin resistance.
1.5 Sucrose Counter Selection of sacB Expression
The E. coli JC9604lacZ strain, generated as described in FIG. 11, was cotransformed with a sacB-neo PCR fragment and pSVpaX1 (Buchholz et al, Nucleic Acids Res. 24 (1996), 4256-4262). After selection on 100 μg/ml ampicillin, 50 μg/ml kanamycin plates, pSVpaX-sacB-neo plasmids were isolated and cotransformed into fresh JC9604lacZ cells with a PCR fragment amplified from pSVpaX1 using primers l*/m*. Oligo m* carried a silent point mutation which generated a BamHI site. Cells were plated on 7% sucrose, 100 μg/ml ampicillin, 40 μg/ml X-gal plates and incubated at 28° C. for 2 days. The blue and white colonies grown on sucrose plates were counted and further checked by restriction analysis.

1.6 Other Methods

DNA preparation and Southern analysis were performed according to standard procedures. Hybridization probes were generated by random priming of fragments isolated from the Tn5 neo gene (PvuII), Hoxa3 gene (both HindIII fragments), lacZ genes (EcoR1 and BamH1 fragments from pSVpaX1), cm gene (BstB1 fragments from pMAK705) and P1 vector fragments (2.2 kb EcoR1 fragments from P1 vector).

2. Results

2.1 Identification of Recombination Events in E. coli
To identify a flexible homologous recombination reaction in E. coli, an assay based on recombination between linear and circular DNAs was designed (FIG. 1, FIG. 3). Linear DNA carrying the Tn5 kanamycin resistance gene (neo) was made by PCR (FIG. 3 a). Initially, the oligonucleotides used for PCR amplification of neo were 60 mers consisting of 42 nucleotides at their 5′ ends identical to chosen regions in the plasmid and, at the 3′ ends, 18 nucleotides to serve as PCR primers. Linear and circular DNAs were mixed in equimolar proportions and co-transformed into a variety of E. coli hosts. Homologous recombination was only detected in sbcA E. coli hosts. More than 95% of double ampicillin/kanamycin resistant colonies (FIG. 3 b) contained the expected homologously recombined plasmid as determined by restriction digestion and sequencing. Only a low background of kanamycin resistance, due to genomic integration of the neo gene, was apparent (not shown).
The linear plus circular recombination reaction was characterized in two ways. The relationship between homology arm length and recombination efficiency was simple, with longer arms recombining more efficiently (FIG. 3 c). Efficiency increased within the range tested, up to 60 bp. The effect of distance between the two chosen homology sites in the recipient plasmid was examined (FIG. 3 d). A set of eight PCR fragments was generated by use of a constant left homology arm with differing right homology arms. The right homology arms were chosen from the plasmid sequence to be 0-3100 bp from the left. Correct products were readily obtained from all, with less than 4 fold difference between them, although the insertional product (O) was least efficient. Correct products also depended on the presence of both homology arms, since PCR fragments containing only one arm failed to work.

2.2 Involvement of RecE and RecT

The relationship between host genotype and this homologous recombination reaction was more systematically examined using a panel of E. coli strains deficient in various recombination components (Table 2).

Table 2

Only the two sbcA strains, JC8679 and JC9604 presented the intended recombination products and RecA was not required. In sbcA strains, expression of RecE and RecT is activated. Dependence on recE can be inferred from comparison of JC8679 with JC8691. Notably no recombination products were observed in JC9387 suggesting that the sbcBC background is not capable of supporting homologous recombination based on 50 nucleotide homology arms.
To demonstrate that RecE and RecT are involved, part of the recET operon was cloned into an inducible expression vector to create pBAD24-recET (FIG. 6 a). the recE gene was truncated at its N-terminal end, as the first 588 a.a.s of RecE are dispensable. The recBC strain, JC5547, was transformed with pBAD24-recET and a time course of RecE/RecT induction performed by adding arabinose to the culture media at various times before harvesting for competent cells. The batches of harvested competent cells were evaluated for protein expression by gel electrophoresis (FIG. 6 b) and for recombination between a linear DNA fragment and the endogenous pBAD24-recET plasmid (FIG. 6 c). Without induction of RecE/RecT, no recombinant products were found, whereas recombination increased in approximate concordance with increased RecE/RecT expression. This experiment also shows that co-transformation of linear and circular DNAs is not essential and the circular recipient can be endogenous in the host. From the results shown in FIGS. 3, 6 and Table 2, we conclude that RecE and RecT mediate a very useful homologous recombination reaction in recBC E. coli at workable frequencies. Since RecE and RecT are involved, we refer to this way of recombining linear and circular DNA fragments as “ET cloning”.

2.3 Application of ET Cloning to Large Target DNAs

To show that large DNA episomes could be manipulated in E. coli, a >76 kb P1 clone that contains at least 59 kb of the intact mouse Hoxa complex, (confirmed by DNA sequencing and Southern blotting), was transferred to an E. coli strain having an sbcA background (JC9604) and subjected to two rounds of ET cloning. In the first round, the Tn903 kanamycin resistance gene resident in the P1 vector was replaced by an ampicillin resistance gene (FIG. 4). In the second round, the interval between the Hoxa3 and a4 genes was targeted either by inserting the neo gene between two base pairs upstream of the Hoxa3 proximal promoter, or by deleting 6203 by between the Hoxa3 and a4 genes (FIG. 8 a). Both insertional and deletional ET cloning products were readily obtained (FIG. 8 b, lanes 2, 3 and 5) showing that the two rounds of ET cloning took place in this large E. coli episome with precision and no apparent unintended recombination.
The general applicability of ET cloning was further examined by targeting a gene in the E. coli chromosome (FIG. 9 a). The β-galactosidase (lacZ) gene of JC9604 was chosen so that the ratio between correct and incorrect recombinants could be determined by evaluating β-galactosidase expression. Standard conditions (0.2 μg PCR fragment; 50 μl competent cells), produced 24 primary colonies, 20 of which were correct as determined by β-galactosidase expression (FIG. 9 b), and DNA analysis (FIG. 9 c, lanes 3-6).

2.4 Secondary Recombination Reactions to Remove Operational Sequences

The products of ET cloning as described above are limited by the necessary inclusion of selectable marker genes. Two different ways to use a further recombination step to remove this limitation were developed. In the first way, site specific recombination mediated by either Flp or Cre recombinase was employed. In the experiments of FIGS. 8 and 9, either Flp recombination target sites (FRTs) or Cre recombination target sites (loxPs) were included to flank the neo gene in the linear substrates. Recombination between the FRTs or loxPs was accomplished by Flp or Cre, respectively, expressed from plasmids with the pSC101 temperature sensitive replication origin (Hashimoto-Gotoh and Sekiguchi, J. Bacteriol. 131 (1977), 405-412) to permit simple elimination of these plasmids after site specific recombination by temperature shift. The precisely recombined Hoxa P1 vector was recovered after both ET and Flp recombination with no other recombination products apparent (FIG. 8, lanes 4 and 6). Similarly, Cre recombinase precisely recombined the targeted lacZ allele (FIG. 9, lanes 7-10). Thus site specific recombination can be readily coupled with ET cloning to remove operational sequences and leave a 34 by site specific recombination target site at the point of DNA manipulation.
In the second way to remove the selectable marker gene, two rounds of ET cloning, combining positive and counter selection steps, were used to leave the DNA product free of any operational sequences (FIG. 10 a).
Additionally this experiment was designed to evaluate, by a functional test based on β-galactosidase activity, whether ET cloning promoted small mutations such as frame shift or point mutations within the region being manipulated. In the first round, the lacZ gene of pSVpaX1 was disrupted with a 3.3 kb PCR fragment carrying the neo and B. subtilis sacB (Blomfield et al., Mol. Microbiol. 5 (1991), 1447-1457) genes, by selection for kanamycin resistance (FIG. 10 a). As shown above for other positively selected recombination products, virtually all selected colonies were white (FIG. 10 b), indicative of successful lacZ disruption, and 17 of 17 were confirmed as correct recombinants by DNA analysis. In the second round, a 1.5 kb PCR fragment designed to repair lacZ was introduced by counter selection against the sacB gene. Repair of lacZ included a silent point mutation to create a BamH1 restriction site. Approximately one quarter of sucrose resistant colonies expressed β-galactosidase, and all analyzed (17 of 17; FIG. 10 c) carried the repaired lacZ gene with the BamH1 point mutation. The remaining three quarters of sucrose resistant colonies did not express β-galactosidase, and all analyzed (17 of 17; FIG. 10 c) had undergone a variety of large mutational events, none of which resembled the ET cloning product. Thus, in two rounds of ET cloning directed at the lacZ gene, no disturbances of R-galactosidase activity by small mutations were observed, indicating the RecE/RecT recombination works with high fidelity. The significant presence of incorrect products observed in the counter selection step is an inherent limitation of the use of counter selection, since any mutation that ablates expression of the counter selection gene will be selected. Notably, all incorrect products were large mutations and therefore easily distinguished from the correct ET product by DNA analysis. In a different experiment (FIG. 5), we observed that ET cloning into pZero2.1 (InVitroGen) by counter selection against the ccdB gene gave a lower background of incorrect products (8%), indicating that the counter selection background is variable according to parameters that differ from those that influence ET cloning efficiencies.
2.5 Transference of ET Cloning Between E. coli Hosts
The experiments shown above were performed in recBC— E. coli hosts since the sbcA mutation had been identified as a suppressor of recBC (Barbour et al., Proc. Natl. Acad. Sci. USA 67 (1970), 128-135; Clark, Genetics 78 (1974), 259-271). However, many useful E. coli strains are recBC+, including strains commonly used for propagation of P1, BAC or PAC episomes. To transfer ET cloning into recBC+strains, we developed pBAD-ETγ and pBAD-αβγ (FIGS. 13 and 14). These plasmids incorporate three features important to the mobility of ET cloning. First, RecBC is the major E. coli exonuclease and degrades introduced linear fragments. Therefore the RecBC inhibitor, Redγ (Murphy, J. Bacteriol. 173 (1991), 5808-5821), was included. Second, the recombinogenic potential of RecE/RecT, or Redα/Redβ, was regulated by placing recE or redα under an inducible promoter. Consequently ET cloning can be induced when required and undesired recombination events which are restricted at other times. Third, we observed that ET cloning efficiencies are enhanced when RecT, or Redβ, but not RecE, or Redα, is overexpressed. Therefore we placed recT, or redβ, under the strong, constitutive, EM7 promoter.
pBAD-ETγ was transformed into NS3145 E. coli harboring the original Hoxa P1 episome (FIG. 11 a). A region in the P1 vector backbone was targeted by PCR amplification of the chloramphenicol resistance gene (cm) flanked by n and p homology arms. As described above for positively selected ET cloning reactions, most (>90%) chloramphenicol resistant colonies were correct. Notably, the overall efficiency of ET cloning, in terms of linear DNA transformed, was nearly three times better using pBAD-ETγ than with similar experiments based on targeting the same episome in the sbcA host, JC9604. This is consistent with our observation that overexpression of RecT improves ET cloning efficiencies.
A comparison between ET cloning efficiencies mediated by RecE/RecT, expressed from pBAD-ETγ, and Redα/Redβ, expressed from pBAD-αβγ was made in the recA−, recBC+E. coli strain, DK1 (FIG. 12). After transformation of E. coli DK1 with either pBAD-ETγ or pBAD-αβγ, the same experiment as described in FIG. 6 a, c, to replace the bla gene of the pBAD vector with a chloramphenicol gene was performed. Both pBAD-ETγ or pBAD-αβγ presented similar ET cloning efficiencies in terms of responsiveness to arabinose induction of RecE and Redα, and number of targeted events.

Claims

1. A method for cloning DNA molecules in procaryotic cells comprising the steps of:

a) providing a procaryotic host cell capable of performing homologous recombination,

b) contacting in said host cell a circular first DNA molecule which is capable of being replicated in said host cell with a second DNA molecule comprising at least two regions of sequence homology to regions on the first DNA molecule, under conditions which favour homologous recombination between said first and second DNA molecules and

c) selecting a host cell in which homologous recombination between said first and second DNA molecules has occurred.

2. The method according to claim 1 wherein the homologous recombination occurs via the recET cloning mechanism.

3. The method according to claim 2 wherein the host cell is capable of expressing recE and recT genes.

4. The method according to claim 3 wherein the recE and recT genes are selected from E. coli recE and recT genes or from λredα and redβ genes.

5. The method according to claim 3 wherein the host cell is transformed with at least one vector capable of expressing recE and/or recT genes.

6. The method of claim 3 wherein the expression of the recE and/or recT genes is under control of a regulatable promoter.

7. The method of claim 5 wherein the recT gene is overexpressed versus the recE gene.

8. The method according to claim 3 wherein the recE gene is selected from a nucleic acid molecule comprising

(a) the nucleic acid sequence from position 1320 (ATG) to 2159 (GAC) as depicted in FIG. 7B,

(b) the nucleic acid sequence from position 1320 (ATG) to 1998 (CGA) as depicted in FIG. 13B,

(c) a nucleic acid encoding the same polypeptide within the degeneracy of the genetic code and/or (d) a nucleic acid sequence which hybridizes under stringent conditions with the nucleic acid sequence from (a), (b) and/or (c).

9. The method according to claim 3 wherein the recT gene is selected from a nucleic acid molecule comprising

(a) the nucleic acid sequence from position 2155 (ATG) to 2961 (GAA) as depicted in FIG. 7B,

(b) the nucleic acid sequence from position 2086 (ATG) to 2868 (GCA) as depicted in FIG. 13B,

(c) a nucleic acid encoding the same polypeptide within the degeneracy of the genetic code and/or (d) a nucleic acid sequence which hybridizes under stringent conditions with the nucleic acid sequences from (a), (b) and/or (c).

10. The method according to claim 1 wherein the host cell is a gram-negative bacterial cell.

11. The method according to claim 10 wherein the host cell is an Escherichia coli cell.

12. The method according to claim 11 wherein the host cell is an Escherichia coli K12 strain.

13. The method according to claim 12 wherein the E. coli strain is selected from JC 8679 and JC 9604.

14. The method according to claim 1 wherein the host cell further is capable of expressing a recBC inhibitor gene.

15. The method according to claim 14 wherein the host cell is transformed with a vector expressing the recBC inhibitor gene.

16. The method according to claim 14 wherein the recBC inhibitor gene is selected from a nucleic acid molecule comprising

(a) the nucleic acid sequence from position 3588 (ATG) to 4002 (GTA) as depicted in FIG. 13B,

(b) a nucleic acid encoding the same polypeptide within the degeneracy of the genetic code and/or

(c) a nucleic acid sequence which hybridizes under stringent conditions (as defined above) with the nucleic acid sequence from (a) and/or (b).

17. The method according to claim 13 wherein the host cell is a prokaryotic recBC+cell.

18. The method according to claim 1 wherein the first DNA molecule is an extrachromosomal DNA molecule containing an origin of replication which is operative in the host cell.

19. The method according to claim 18 wherein the first DNA molecule is selected from plasmids, cosmids, P1 vectors, BAC vectors and PAC vectors.

20. The method according to claim 1 wherein the first DNA molecule is a host cell chromosome.

21. The method according to claim 1 wherein the second DNA molecule is linear.

22. The method according to claim 1 wherein the regions of sequence homology are at least 15 nucleotides each.

23. The method according to claim 1 wherein the second DNA molecule is obtained by an amplification reaction.

24. The method according to claim 1 wherein the first and/or second DNA molecules are introduced into the host cells by transformation.

25. The method according to claim 24 wherein the transformation method is electroporation.

26. The method according to claim 1 wherein the first and second DNA molecules are introduced into the host cell simultaneously by co-transformation.

27. The method according to claim 1 wherein the second DNA molecule is introduced into a host cell in which the first DNA molecule is already present.

28. The method according to claim 1 wherein the second DNA molecule contains at least one marker gene placed between the two regions of sequence homology and wherein homologous recombination is detected by expression of said marker gene.

29. The method according to claim 28 wherein gene presence is selected from antibiotic resistance genes, deficiency complementation genes and reporter genes.

30. The method of claim 1 wherein the first DNA molecule contains at least one marker gene between the two regions of sequence homology and wherein homologous recombination is detected by lack of expression of said marker gene.

31. The method of claim 1 wherein said marker gene is selected from genes which, under selected conditions, convey a toxic or bacteriostatic effect on the cell, and reporter genes.

32. A method according to claim 1 wherein the first DNA molecule contains at least one target site for a site specific recombinase between the two regions of sequence homology and wherein homologous recombination is detected by removal of said target site.

33. A method for cloning DNA molecules comprising the steps of:

(a) providing a source of RecE and RecT proteins,

(b) contacting a first DNA molecule which is capable of being replicated in a suitable host cell with a second DNA molecule comprising at least two regions of sequence homology to regions on the first DNA molecule, under conditions which favour homologous recombination between said first and second DMA molecules and

(c) selecting DNA molecules in which homologous recombination between said first and second DNA molecules has occurred.

34. The method of claim 33 wherein said RecE and RecT or proteins are selected from E. coli RecE and RecT proteins or from phage λ Redα and Redβ proteins.

35. The method of claim 33 wherein the recombination occurs in vitro.

36. The method of claim 33 wherein the recombination occurs in vivo.

37. A method for making a recombinant DNA molecule comprising introducing into a prokaryotic host cell a circular first DNA molecule which is capable of being replicated in said host cell, and introducing a second DNA molecule comprising a first and a second region of sequence homology to a third and fourth region, respectively, on the first DNA molecule, said host cell being capable of performing homologous recombination, such that a recombinant DNA molecule is made, said recombinant DNA molecule comprising the first DNA molecule wherein the sequences between said third and fourth regions have been replaced by sequences between the first and second regions of the second DNA molecule.

38. The method according to claim 37 which further comprises detecting the recombinant DNA molecule.

39. A method for making a recombinant DNA molecule comprising introducing into a prokaryotic host cell, containing a chromosomal first DNA molecule, a second DNA molecule comprising a first and a second region of sequence homology to a third and a fourth region, respectively, on the host chromosomal first DNA molecule, said host cell being capable of performing homologous recombination, such that a recombinant DNA molecule is made, said recombinant DNA molecule comprising the chromosomal first DNA molecule wherein the sequences between said third and fourth regions have been replaced by sequences between the first and second regions of the second DNA molecule.

40. The method according to claim 39 which further comprises detecting the recombinant DNA molecule.

41. The method according to claim 37, wherein the host cell is capable of expressing RecE and RecT proteins or λ Redα and λRedβ proteins.

42. A method for cloning DNA molecules comprising the steps of:

(a) contacting in vitro a first DNA molecule with a second DNA molecule comprising at least two regions of sequence homology to regions on the first DNA molecule, in the presence of RecE and RecT proteins and under conditions which favour homologous recombination between said first and second DNA molecules; and

(b) selecting a DNA molecule in which homologous recombination between said first and second DNA molecules has occurred.

43. A method for making a recombinant DNA molecule comprising contacting in vitro a first DNA molecule with a second DNA molecule comprising a first and a second region of sequence homology to a third and a fourth region on the first DNA molecule, in the presence of RecE and RecT proteins and under conditions in which homologous recombination can occur, such that a recombinant DNA molecule is made, said recombinant DNA molecule comprising the first DNA molecule wherein the sequences between said third and fourth regions have been replaced by sequences between the first and second regions of the second DNA molecule.

44. The method of claim 42, which further comprises between steps (a) and (b) the step of introducing the product step (a) into a cell, wherein recombination occurs in the cell.

45. Use of cells capable of expressing the recE and recT genes as a host cell for a cloning method involving homologous recombination.

46. Use of a vector system capable of expressing recE and recT genes in a host cell for a cloning method involving homologous recombination.

47. Use of claim 45 wherein the recE and recT genes are selected from E. coli recE and recT genes or from A reda and redβ genes.

48. Use of a source of RecE and RecT proteins for a cloning method involving homologous recombination.

49. Use of claim 48 wherein said RecE and RecT or proteins are selected from E. coli RecE and RecT proteins or from phage A Reda and Redβ proteins.

50. A reagent kit for cloning comprising (a) a host cell (b) means of expressing recE and recT genes in said host cell and (c) a recipient cloning vehicle capable of being replicated in said cell.

51. The reagent kit according to claim 50 wherein the means (b) comprise a vector system capable of expressing the recE and recT genes in the host cell.

52. The reagent kit according to claim 50 wherein the recE and recT genes are selected from E. coli recE and recT genes or from A reda and redβ genes.

53. A reagent kit for cloning comprising (a) a source for Re E and RecT proteins and (b) a recipient cloning vehicle capable of being propagated in a host cell.

54. The reagent kit according to claim 53 further comprising a host cell suitable for propagating said recipient cloning vehicle.

55. The reagent kit according to claim 53 wherein said RecE and RecT or proteins are selected from E. coli RecE and RecT proteins or from phage λ Redα and Redβ proteins.

56. The reagent kit according to claim 50 further comprising means for expressing a site specific recombinase in said host cell.

57. The reagent kit according to claim 50 further comprising nucleic acid amplification primers comprising a region of homology to said recipient cloning vehicle.

58. A reagent kit for cloning comprising first and second DNA amplification primers and a recipient cloning vehicle that is a circular DNA molecule, said first DNA amplification primer having a first region of sequence homology to a third region on the circular recipient cloning vehicle, and said second DNA amplification primer having a second region of sequence homology to a fourth region on the circular recipient cloning vehicle.

59. The reagent kit of claim 58, further comprising a prokaryotic host cell that is capable of performing homologous recombination.

60. The reagent kit of claim 58, further comprising a means of expressing RecE and RecT proteins or Redα and Redβ proteins.

61. The reagent kit according to claim 58, wherein the means comprises a vector system capable of expressing the recE and recT genes in the host cell.

62. The reagent kit according to claim 58, further comprising a phenotypic marker located in the recipient cloning vehicle between the third and fourth regions of sequence homology.

63. The reagent kit according to claim 58, wherein the recipient cloning vehicle further comprises a recognition site for a site-specific recombinase on the recipient cloning vehicle between the third and fourth regions of sequence homology.

64. The reagent kit of claim 63, further comprising means for expressing a site-specific recombinase in said host cell.