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 genetically engineered prokaryotic cell comprising a homologous recombinant DNA molecule made by homologous recombination between a circular first molecule and a linear second DNA molecule, and prepared by a method comprising the following steps:
 a) providing a prokaryotic host cell capable of performing homologous recombination, wherein the host cell expresses recE and recT genes;   b) contacting in said host cell
 said circular first DNA molecule which is capable of being replicated in said host cell, said first DNA molecule being the host cell chromosome, with 
 said linear second DNA molecule comprising at least two regions of sequence homology to regions on the first DNA molecule and further comprising a DNA fragment to be integrated into the first DNA molecule, 
 under conditions which favor homologous recombination between said first and second DNA molecules; 
 wherein when said homologous recombination occurs, it is mediated by gene products of said recE and recT genes; and 
   c) selecting a host cell in which homologous recombination between said first and second DNA molecules has occurred, thereby obtaining the genetically engineered prokaryotic cell.   
     
     
         2 . A genetically engineered prokaryotic cell comprising a homologous recombinant DNA molecule made by homologous recombination between a circular first molecule and a linear second DNA molecule and free of at least one marker gene, and prepared by a method comprising the following steps:
 a) providing a prokaryotic host cell capable of performing homologous recombination, wherein the host cell expresses recE and recT genes;   b) contacting in said host cell
 said circular first DNA molecule which is capable of being replicated in said host cell, said first DNA molecule being the host cell chromosome, with 
 said linear second DNA molecule comprising at least two regions of sequence homology to regions on the first DNA molecule and further comprising a DNA fragment to be integrated into the first DNA molecule, wherein the second DNA molecule contains said at least one marker gene placed between the two regions of sequence homology, 
 under conditions which favor homologous recombination between said first and second DNA molecules, wherein when said homologous recombination occurs, it is mediated by gene products of said recE and recT genes; 
   c) selecting a host cell in which homologous recombination between said first and second DNA molecules has occurred,
 wherein homologous recombination is detected by expression of said at least one marker gene; and 
   d) removing said at least one marker gene from a DNA molecule generated by the homologous recombination between the first and second DNA molecules in said host cell of step c), thereby obtaining the genetically engineered prokaryotic cell.   
     
     
         3 . The prokaryotic cell according to  claim 1  or  2 , wherein the recE and recT genes are selected from λredα and redβ genes or from  E. coli  recE and recT genes. 
     
     
         4 . The prokaryotic cell according to  claim 1  or  2 , wherein the host cell comprises at least one vector capable of expressing recE and/or recT genes. 
     
     
         5 . The prokaryotic cell according to  claim 1  or  2 , wherein the expression of the recE and/or recT genes is under control of a regulatable promoter. 
     
     
         6 . The prokaryotic cell according to  claim 1  or  2 , wherein the recE gene is selected from a nucleic acid molecule comprising
 (a) the nucleic acid sequence from position 1320 (ATG) to 2159 (GAC) of SEQ ID NO.: 2, 
 (b) the nucleic acid sequence from position 1320 (ATG) to 1998 (CGA) of SEQ ID NO.: 10, 
 (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). 
 
     
     
         7 . The prokaryotic cell according to  claim 1  or  2 , wherein the recT gene is selected from a nucleic acid molecule comprising
 (a) the nucleic acid sequence from position 2155 (ATG) to 2961 (GAA) of SEQ ID NO.: 4, 
 (b) the nucleic acid sequence from position 2086 (ATG) to 2868 (GCA) of SEQ ID NO.: 10, 
 (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). 
 
     
     
         8 . The prokaryotic cell according to  claim 1  or  2 , wherein the host cell is a gram-negative bacterial cell. 
     
     
         9 . The prokaryotic cell according to  claim 8  wherein the host cell is an  Escherichia coli  cell. 
     
     
         10 . The prokaryotic cell according to  claim 9  wherein the host cell is an  Escherichia coli  K12 strain. 
     
     
         11 . The prokaryotic cell according to  claim 1  or  2 , wherein the host cell further is capable of expressing a recBC inhibitor gene. 
     
     
         12 . The prokaryotic cell according to  claim 11  wherein the host cell comprises a vector expressing the recBC inhibitor gene. 
     
     
         13 . The prokaryotic cell according to  claim 11  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) of SEQ ID NO.: 10, 
 (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 with the nucleic acid sequence from (a) and/or (b). 
 
     
     
         14 . The prokaryotic cell according to  claim 1  or  2 , wherein the regions of sequence homology are at least 15 nucleotides each. 
     
     
         15 . The prokaryotic cell according to  claim 1  or  2 , wherein the second DNA molecule is obtained by an amplification reaction. 
     
     
         16 . The prokaryotic cell according to  claim 1  or  2 , wherein the second DNA molecule is introduced into the host cells by transformation. 
     
     
         17 . The prokaryotic cell according to  claim 16  wherein the transformation method is electroporation. 
     
     
         18 . The prokaryotic cell 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. 
     
     
         19 . The prokaryotic cell according to  claim 18 , wherein said marker gene on the second DNA molecule is selected from antibiotic resistance genes, deficiency complementation genes and reporter genes. 
     
     
         20 . The prokaryotic cell according to  claim 2 , wherein said marker gene on the second DNA molecule is selected from antibiotic resistance genes, deficiency complementation genes and reporter genes. 
     
     
         21 . The prokaryotic cell according to  claim 1  or  2 , 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. 
     
     
         22 . The prokaryotic cell according to  claim 21  wherein said marker gene on the first DNA molecule is selected from genes which, under selected conditions, convey a toxic or bacteriostatic effect on the cell, and reporter genes. 
     
     
         23 . The prokaryotic cell according to  claim 1  or  2 , 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. 
     
     
         24 . The prokaryotic cell according to  claim 1 , wherein the chromosome of the prokaryotic cell obtained in step c) differs from the chromosome of the prokaryotic host cell of step a) by at least one mutation selected from the group consisting of point mutations, insertions, and deletions. 
     
     
         25 . The prokaryotic cell according to  claim 2 , wherein the chromosome of the prokaryotic cell obtained in step d) differs from the chromosome of the prokaryotic host cell of step a) by at least one mutation selected from the group consisting of point mutations, insertions, and deletions. 
     
     
         26 . A method for preparing a genetically engineered prokaryotic cell comprising the steps of:
 a) providing a prokaryotic host cell capable of performing homologous recombination, wherein the host cell expresses recE and recT genes;   b) contacting in said host cell
 a circular first DNA molecule which is capable of being replicated in said host cell, said first DNA molecule being the host cell chromosome, with 
 a linear second DNA molecule comprising at least two regions of sequence homology to regions on the first DNA molecule and further comprising a DNA fragment to be integrated into the first DNA molecule, 
 under conditions which favor homologous recombination between said first and second DNA molecules, wherein when said homologous recombination occurs, it is mediated by gene products of said recE and recT genes; and 
   c) selecting a host cell in which homologous recombination between said first and second DNA molecules has occurred, thereby obtaining a genetically engineered prokaryotic cell.   
     
     
         27 . The method according to  claim 26  wherein the recE and recT genes are selected from λredα and redβ genes or from  E. coli  recE and recT genes. 
     
     
         28 . The method according to  claim 26  wherein the host cell is transformed with at least one vector capable of expressing recE and/or recT genes. 
     
     
         29 . The method of  claim 26  wherein the expression of the recE and/or recT genes is under control of a regulatable promoter. 
     
     
         30 . The method according to  claim 26  wherein the recE gene is selected from a nucleic acid molecule comprising
 (a) the nucleic acid sequence from position 1320 (ATG) to 2159 (GAC) of SEQ ID NO.: 2, 
 (b) the nucleic acid sequence from position 1320 (ATG) to 1998 (CGA) of SEQ ID NO.: 10, 
 (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). 
 
     
     
         31 . The method according to  claim 26  wherein the recT gene is selected from a nucleic acid molecule comprising
 (a) the nucleic acid sequence from position 2155 (ATG) to 2961 (GAA) of SEQ ID NO.: 4, 
 (b) the nucleic acid sequence from position 2086 (ATG) to 2868 (GCA) of SEQ ID NO.: 10, 
 (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). 
 
     
     
         32 . The method according to  claim 26  wherein the host cell is a gram-negative bacterial cell. 
     
     
         33 . The method according to  claim 32  wherein the host cell is an  Escherichia coli  cell. 
     
     
         34 . The method according to  claim 33  wherein the host cell is an  Escherichia coli  K12 strain. 
     
     
         35 . The method according to  claim 26  wherein the host cell further is capable of expressing a recBC inhibitor gene. 
     
     
         36 . The method according to  claim 35  wherein the host cell is transformed with a vector expressing the recBC inhibitor gene. 
     
     
         37 . The method according to  claim 35  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) of SEQ ID NO.: 10, 
 (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 with the nucleic acid sequence from (a) and/or (b). 
 
     
     
         38 . The method according to  claim 26  wherein the regions of sequence homology are at least 15 nucleotides each. 
     
     
         39 . The method according to  claim 26  wherein the second DNA molecule is obtained by an amplification reaction. 
     
     
         40 . The method according to  claim 26  wherein the second DNA molecule is introduced into the host cells by transformation. 
     
     
         41 . The method according to  claim 40  wherein the transformation method is electroporation. 
     
     
         42 . The method according to  claim 26  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. 
     
     
         43 . The method according to  claim 42  wherein said marker gene on the second DNA molecule is selected from antibiotic resistance genes, deficiency complementation genes and reporter genes. 
     
     
         44 . The method according to  claim 43  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. 
     
     
         45 . The method of  claim 44  wherein said marker gene on the first DNA molecule is selected from genes which, under selected conditions, convey a toxic or bacteriostatic effect on the cell, and reporter genes. 
     
     
         46 . The method according to  claim 26  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. 
     
     
         47 . The method according to  claim 26  wherein the chromosome of the prokaryotic cell obtained in step c) differs from the chromosome of the prokaryotic host cell of step a) by at least one mutation selected from the group consisting of point mutations, insertions, and deletions. 
     
     
         48 . The method of  claim 42 , further comprising the step of:
 d) removing said at least one marker gene from a DNA molecule generated by the homologous recombination between the first and second DNA molecules in said host cell of step c), thereby obtaining a genetically engineered prokaryotic cell.   
     
     
         49 . The method according to  claim 48 , wherein the chromosome of the prokaryotic cell obtained in step d) differs from the chromosome of the prokaryotic host cell of step a) by at least one mutation selected from the group consisting of point mutations, insertions, and deletions.