Compositions and methods for recombinational cloning of nucleic acid molecules

Abstract

The present invention relates generally to compositions and methods for enhancing recombinational cloning of nucleic acid molecules. In particular, the invention relates to compositions comprising one or more ribosomal proteins and one or more additional protein components required for recombinational cloning. More particularly, the invention relates to such compositions wherein the ribosomal proteins are one or more E. coli ribosomal proteins, still more particularly wherein the ribosomal proteins are selected from the group of E. coli ribosomal proteins consisting of S10, S14, S15, S16, S17, S18, S19, S20, S21, L20, L21, and L23 through L34, and most particularly S20, L27, and S15. The invention also relates to the use of these compositions in methods for recombinational cloning of nucleic acids, in vitro and in vivo, to provide chimeric DNA molecules that have particular characteristics and/or DNA segments. The invention also relates to isolated nucleic acid molecules produced by the methods of the invention, to vectors comprising such nucleic acid molecules, and to host cells comprising such nucleic acid molecules and vectors.

1 . An in vitro composition comprising:
 (a) at least one ribosomal protein present in an amount sufficient to enhance recombinational cloning,   (b) at least one recombinational protein present in an amount sufficient to facilitate recombinational cloning, and   (c) a vector comprising at least one recombination site and a toxic gene.   
     
     
         2 . The composition of  claim 1 , wherein said toxic gene is selected from the group consisting of ccdB, GATA-1, and kicB. 
     
     
         3 . The composition of  claim 1 , wherein said at least one ribosomal protein is a prokaryotic ribosomal protein. 
     
     
         4 . The composition of  claim 1 , wherein said at least one ribosomal protein is an  Escherichia coli  ribosomal protein. 
     
     
         5 . The composition of  claim 1 , wherein said at least one ribosomal protein is a basic ribosomal protein. 
     
     
         6 . The composition of  claim 1 , wherein said at least one ribosomal protein has a molecular weight of less than about 14 kilodaltons. 
     
     
         7 . The composition of  claim 4 , wherein said  Escherichia coli  ribosomal protein is selected from the group of  Escherichia coli  ribosomal proteins consisting of S10, S14, S15, S16, S17, S18, S19, S20, S21, L21, L23, L24, L25, L27, L28, L29, L30, L31, L32, L33 and L34. 
     
     
         8 . The composition of  claim 4 , wherein said  Escherichia coli  ribosomal protein is S20. 
     
     
         9 . The composition of  claim 4 , wherein said  Escherichia coli  ribosomal protein is L27. 
     
     
         10 . The composition of claim  68 , wherein said  Escherichia coli  ribosomal protein is S15. 
     
     
         11 . The composition of  claim 1 , wherein said at least one recombination protein is a prokaryotic recombination protein. 
     
     
         12 . The composition of  claim 1 , wherein said at least one recombination protein is selected from the group consisting of Int, Cre, FLP, Xis, IHF, FIS and HU, and combinations thereof. 
     
     
         13 . The composition of  claim 1 , wherein said at least one recombination protein is Int. 
     
     
         14 . The composition of  claim 1 , further comprising one or more nucleic acid molecules selected from the group consisting of one or more Insert Donor molecules, one or more Vector Donor molecules, one or more Cointegrate molecules, one or more Product molecules, and one or more Byproduct molecules. 
     
     
         15 . A kit comprising the composition of  claim 1 . 
     
     
         16 . The kit of  claim 15 , which further comprises one or more polymerases. 
     
     
         17 . The kit of  claim 15 , which further comprises one or more restriction endonucleases. 
     
     
         18 . An in vitro composition comprising:
 (a) at least one ribosomal protein present in an amount sufficient to enhance recombinational cloning,   (b) at least one recombinational protein present in an amount sufficient to facilitate recombinational cloning; and   (c) a vector comprising at least two recombination sites selected from the group consisting of two attB sites, two attP sites, two attL sites, and two attR sites.   
     
     
         19 . The composition of  claim 18 , wherein said at least two recombination sites are two attB sites. 
     
     
         20 . The composition of  claim 18 , wherein said at least two recombination sites are two attP sites. 
     
     
         21 . The composition of  claim 18 , wherein said at least two recombination sites are two attL sites. 
     
     
         22 . The composition of  claim 18 , wherein said at least two recombination sites are two attR sites. 
     
     
         23 . The composition of  claim 18 , wherein said at least one ribosomal protein is a prokaryotic ribosomal protein. 
     
     
         24 . The composition of  claim 18 , wherein said at least one ribosomal protein is an  Escherichia coli  ribosomal protein. 
     
     
         25 . The composition of  claim 18 , wherein said at least one ribosomal protein is a basic ribosomal protein. 
     
     
         26 . The composition of  claim 18 , wherein said at least one ribosomal protein has a molecular weight of less than about 14 kilodaltons. 
     
     
         27 . The composition of  claim 24  wherein said  Escherichia coli  ribosomal protein is selected from the group of  Escherichia coli  ribosomal proteins consisting of S10, S14, S15, S16, S17, S18, S19, S20, S21, L21, L23, L24, L25, L27, L28, L29, L30, L31, L32, L33 and L34. 
     
     
         28 . The composition of  claim 24 , wherein said  Escherichia coli  ribosomal protein is S20. 
     
     
         29 . The composition of  claim 24 , wherein said  Escherichia coli  ribosomal protein is L27. 
     
     
         30 . The composition of  claim 24 , wherein said  Escherichia coli  ribosomal protein is S15. 
     
     
         31 . The composition of  claim 18 , wherein said at least one recombination protein is a prokaryotic recombination protein. 
     
     
         32 . The composition of  claim 18 , wherein said at least one recombination protein is selected from the group consisting of Int, Cre, FLP, Xis, IHF, FIS and HU, and combinations thereof. 
     
     
         33 . The composition of  claim 18 , wherein said at least one recombination protein is Int. 
     
     
         34 . The composition of  claim 18 , further comprising one or more nucleic acid molecules selected from the group consisting of one or more Insert Donor molecules, one or more Vector Donor molecules, one or more Cointegrate molecules, one or more Product molecules, and one or more Byproduct molecules. 
     
     
         35 . A kit comprising the composition of  claim 18 . 
     
     
         36 . The kit of  claim 35 , which further comprises one or more polymers. 
     
     
         37 . The kit of  claim 35 , which further comprises one or more restriction endonucleases. 
     
     
         38 . An in vitro composition comprising:
 (a) at least one ribosomal protein present in an amount sufficient to enhance recombinational cloning,   (b) at least one recombinational protein present in an amount sufficient to facilitate recombinational cloning; and   (c) a nucleic acid molecule comprising a promoter which allows for transcription in an animal cell.   
     
     
         39 . The composition of  claim 38 , wherein said nucleic acid molecule is a CMV promoter. 
     
     
         40 . The composition of  claim 38 , wherein said at least one ribosomal protein is a prokaryotic ribosomal protein. 
     
     
         41 . The composition of  claim 38 , wherein said at least one ribosomal protein is an  Escherichia coli  ribosomal protein. 
     
     
         42 . The composition of  claim 38 , wherein said at least one ribosomal protein is a basic ribosomal protein. 
     
     
         43 . The composition of  claim 38 , wherein said at least one ribosomal protein has a molecular weight of less than about 14 kilodaltons. 
     
     
         44 . The composition of  claim 41 , wherein said  Escherichia coli  ribosomal protein is selected from the group of  Escherichia coli  ribosomal proteins consisting of S10, S14, S15, S16, S17, S18, S19, S20, S21, L21, L23, L24, L25, L27, L28, L29, L30, L31, L32, L33 and L34. 
     
     
         45 . The composition of  claim 41 , wherein said  Escherichia coli  ribosomal protein is S20. 
     
     
         46 . The composition of  claim 41 , wherein said  Escherichia coli  ribosomal protein is L27. 
     
     
         47 . The composition of  claim 41 , wherein said  Escherichia coli  ribosomal protein is S15. 
     
     
         48 . The composition of  claim 38 , wherein said at least one recombination protein is a prokaryotic recombination protein. 
     
     
         49 . The composition of  claim 38 , wherein said at least one recombination protein is selected from the group consisting of Int, Cre, FLP, Xis, IHF, FIS and HU, and combinations thereof. 
     
     
         50 . The composition of  claim 38 , wherein said at least one recombination protein is Int. 
     
     
         51 . The composition of  claim 38 , further comprising one or more nucleic acid molecules selected from the group consisting of one or more Insert Donor molecules, one or more Vector Donor molecules, one or more Cointegrate molecules, one or more Product molecules, and one or more Byproduct molecules. 
     
     
         52 . A kit comprising at least one container containing the composition of  claim 1 . 
     
     
         53 . A kit comprising at least one container containing the composition of  claim 18 . 
     
     
         54 . A kit comprising at least one container containing the composition of  claim 38 .