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

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.

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.