title: Recombineering: genetic engineering in bacteria using homologous recombination.
abstract: The bacterial chromosome and bacterial plasmids can be engineered in vivo by homologous recombination using PCR products and synthetic oligonucleotides as substrates. This is possible because bacteriophage-encoded recombination proteins efficiently recombine sequences with homologies as short as 35 to 50 bases. Recombineering allows DNA sequences to be inserted or deleted without regard to location of restriction sites. This unit first describes preparation of electrocompetent cells expressing the recombineering functions and their transformation with dsDNA or ssDNA. It then presents support protocols that describe several two-step selection/counter-selection methods of making genetic alterations without leaving any unwanted changes in the targeted DNA, and a method for retrieving onto a plasmid a genetic marker (cloning by retrieval) from the Escherichia coli chromosome or a co-electroporated DNA fragment. Additional protocols describe methods to screen for unselected mutations, removal of the defective prophage from recombineering strains, and other useful techniques.
authors.affiliation: Basic Science Program, GRCBL-Molecular Control & Genetics Section, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc, Frederick, Maryland.
authors.forename: Lynn C
authors.initials: LC
authors.lastname: Thomason
authors.affiliation: Molecular Control and Genetics Section, Gene Regulation and Chromosome Biology, National Cancer Institute at Frederick, National Institutes of Health, Frederick, Maryland.
authors.forename: James A
authors.initials: JA
authors.lastname: Sawitzke
authors.affiliation: Molecular Control and Genetics Section, Gene Regulation and Chromosome Biology, National Cancer Institute at Frederick, National Institutes of Health, Frederick, Maryland.
authors.forename: Xintian
authors.initials: X
authors.lastname: Li
authors.affiliation: Molecular Control and Genetics Section, Gene Regulation and Chromosome Biology, National Cancer Institute at Frederick, National Institutes of Health, Frederick, Maryland.
authors.forename: Nina
authors.initials: N
authors.lastname: Costantino
authors.affiliation: Molecular Control and Genetics Section, Gene Regulation and Chromosome Biology, National Cancer Institute at Frederick, National Institutes of Health, Frederick, Maryland.
authors.forename: Donald L
authors.initials: DL
authors.lastname: Court
mesh_terms: D004269:DNA, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D059765:Homologous Recombination; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D004269:DNA, Bacterial
keywords: Rac prophage; RecET; bacteria; bacteriophage λ; homologous recombination; recombineering; selection/counter-selection; λ Red system
references.citation: Adhya, S. and Gottesman, M. 1978. Control of transcription termination. Ann. Rev. Biochem. 47:967-996.
references.citation: Alper, M.D. and Ames, B.N. 1975. Positive selection of mutants with deletions of the gal-chl region of the Salmonella chromosome as a screening procedure for mutagens that cause deletions. J. Bacteriol. 121:259-266.
references.citation: Altschul, S.F. , Gish, W. , Miller, W. , Myers, E.W. , and Lipman, D.J. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403-410.
references.citation: Arber, W. , Enquist, L. , Hohn, B. , Murray, N.E. , and Murray, K. 1983. Experimental methods for use with lambda. In Lambda II ( R. Hendrix , J. Roberts , F. Stahl , and R. Weisberg , eds.) pp. 433-471. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
references.citation: Bird, A.W. , Erler, A. , Fu, J. , Hériché, J-K. , Maresca, M. , Zhang, Y. , Hyman, A.A. , and Stewart. A.F. 2011. High-efficiency counterselection recombineering for site-directed mutagenesis in bacterial artificial chromosomes. Nat. Methods 9:103-109.
references.citation: Blomfield, I.C. , Vaughn, V. , Rest, R.F. , and Eisenstein, B.I. 1991. Allelic exchange in Escherichia coli using the Bacillus subtilis sacB gene and a temperature-sensitive pSC101 replicon. Mol. Microbiol. 5:1447-1457.
references.citation: Bochner, B.R. , Huang, H.C. , Schieven, G.L. , and Ames, B.N. 1980. Positive selection for loss of tetracycline resistance. J. Bacteriol. 143:926-933.
references.citation: Bubuenenko, M. , Baker, T. , and Court, D.L. 2007. Essentiality of ribosomal and transcription antitermination proteins analyzed by systematic gene replacement in Escherichia coli . J. Bacteriol. 189:2844-2853.
references.citation: Carter, D.M. and Radding, C.M. 1971. The role of exonuclease and β protein of phage λ in genetic recombination. II. Substrate specificity and the mode of action of λ exonuclease. J. Biol. Chem. 246:2502-2512.
references.citation: Cha, R.S. , Zarbl, H. , Keohavong, P. , and Thilly, W.G. 1992. Mismatch amplification mutation assay (MAMA): Application to the c-H-ras gene. PCR Methods Appl. 2:14-20.
references.citation: Cohen, A. and Clark, A. J. 1986. Synthesis of linear plasmid multimers in Escherichia coli K-12. J. Bacteriol. 167:327-335.
references.citation: Copeland, N.G. , Jenkins, N.A. , and Court, D.L. 2001. Recombineering: A powerful new tool for mouse functional genomics. Nat. Rev. Genet. 2:769-779.
references.citation: Costantino, N. and Court, D.L. 2003. Enhanced levels of lambda Red-mediated recombinants in mismatch repair mutants. Proc. Natl. Acad. Sci. U.S.A. 100:15748-15753.
references.citation: Court, D.L. , Sawitzke, J.A. , and Thomason, L.C. 2002. Genetic engineering using homologous recombination. Ann. Rev. Genet. 36:361-388.
references.citation: Court, D.L. , Swaminathan, S. , Yu, D. , Wilson, H. , Baker, T. , Bubunenko, M. , Sawitzke, J. , and Sharan, S.K. 2003. Mini-lambda: A tractable system for chromosome and BAC engineering. Gene 315:63-69.
references.citation: Datsenko, K.A. and Wanner, B.L. 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U.S.A. 97:6640-6645.
references.citation: Datta, S. , Costantino, N. , and Court, D.L. 2006. A set of recombineering plasmids for Gram-negative bacteria. Gene 379:109-115.
references.citation: Datta, S. , Costantino, N. , Zhou, X. , and Court, D.L. 2008. Identification and analysis of recombineering functions from Gram-negative and Gram-positive bacteria and their phages. Proc. Natl. Acad. Sci. U.S.A. 105:1626-1631.
references.citation: DeVito, J.A. 2008. Recombineering with tolC as a selectable/counter-selectable marker: Remodeling the rRNA operons of Escherichia coli . Nucleic Acids Res. 36:e4.
references.citation: Ellis, H.M. , Yu, D. , DiTizio, T. , and Court, D.L. 2001. High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides. Proc. Natl. Acad. Sci. U.S.A. 98:6742-6746.
references.citation: Fu, J. , Bian, X. , Hu, S. , Wang, H. , Huang, F. , Seibert, P. , Plaza, A. , Xia, L. , Müller, R. , Stewart, A.F. , and Zhang, Y. 2012. Full-length RecE enhances linear-linear homologous recombination and facilitates direct cloning for bioprospecting. Nat. Biotechnol. 30:440-446.
references.citation: Gay, P. , Le Coq, D. , Steinmetz, M. , Berkelman, T. , and Kado, C.I. 1985. Positive selection procedure for entrapment of insertion sequence elements in Gram-negative bacteria. J. Bacteriol. 164:918-921.
references.citation: Hall, S.D. , Kane, M.F. , and Kolodner, R. 1993. Identification and characterization of the Escherichia coli RecT protein, a protein encoded by the recE region that promotes renaturation of homologous single-stranded DNA. J. Bacteriol. 175:277-287.
references.citation: Jiang, W. , Bikard, D. , Cox, D. , Zhang, F. , and Marraffini, L.A. 2013. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat. Biotechnol. 31:233-239.
references.citation: Joseph, J.W. and Kolodner, R. 1983. Exonuclease VIII of Escherichia coli. II. Mechanism of action. J. Biol. Chem. 258:10418-10424.
references.citation: Karu, A.E. , Sakaki, Y. , Echols, H. , and Linn, S. 1975. The γ protein specified by bacteriophage λ. Structure and inhibitory activity for the RecBC enzyme of Escherichia coli . J. Biol. Chem. 250:7377-7387.
references.citation: Kulkarni, S.K. and Stahl, F.W. 1989. Interaction between the sbcC gene of Escherichia coli and the gam gene of phage λ. Genetics 123:249-253.
references.citation: Lee, E.C. , Yu, D. , Martinez de Velasco, J. , Tessarollo, L. , Swing, D.A. , Court, D.L. , Jenkins, N.A. , and Copeland, N.G. 2001. A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA. Genomics 73:56-65.
references.citation: Li, X. , Thomason, L.C. , Sawitzke, J.A. , Costantino, N. , and Court, D.L. 2013. Positive and negative selection using the tetA-sacB cassette: Recombineering and P1 transduction in Escherichia coli . Nucleic Acids Res. 41:e204.
references.citation: Little, J.W. 1967. An exonuclease induced by bacteriophage λ. II. Nature of the enzymatic reaction. J. Biol. Chem. 242:679-686.
references.citation: Maloy, S.R. and Nunn, W.D. 1981. Selection for loss of tetracycline resistance by Escherichia coli . J. Bacteriol. 145:1110-1111.
references.citation: Maresca, M. , Erler, A. , Fu, J. , Friedrich, A. , Zhang, Y. , and Stewart, A.F. 2010. Single-strand heteroduplex intermediates in λ Red homologous recombination. BMC Mol. Biol. 11:54.
references.citation: Miller, J.M. 1972. Generalized transduction: Use of P1 in strain construction. In Experiments in Molecular Genetics ( J.M. Miller , ed.) pp. 201-205. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
references.citation: Mosberg, J.A. , Lajoie, M.J. , and Church, G.M. 2010. Lambda Red recombineering in Escherichia coli occurs through a fully single-stranded intermediate. Genetics 186:791-799.
references.citation: Muniyappa, K. and Radding, C.M. 1986. The homologous recombination system of phage λ. Pairing activities of β protein. J. Biol. Chem. 261:7472-7478.
references.citation: Murphy, K.C. 2012. Phage recombinases and their applications. Adv. Virus Res. 83:367-414.
references.citation: Murphy, K.C. and Campellone, K.G. 2003. Lambda Red-mediated recombinogenic engineering of enterohemorrhagic and enteropathogenic E. coli . BMC Mol. Biol. 4:11.
references.citation: Murphy, K.C. , Campellone, K.G. , and Poteete, A.R. 2000. PCR-mediated gene replacement in Escherichia coli . Gene 246:321-330.
references.citation: Muyrers, J.P.P. , Zhang, Y. , Testa, G. , and Stewart, A.F. 1999. Rapid modification of bacterial artificial chromosomes by ET-recombination. Nucleic Acids Res. 27:1555-1557.
references.citation: Noirot, P. and Kolodner, R.D. 1998. DNA strand invasion promoted by Escherichia coli RecT protein. J. Biol. Chem. 273:12274-12280.
references.citation: Oppenheim, A.B. , Rattray, A.J. , Bubunenko, M. , Thomason, L.C. , and Court, D.L. 2004. In vivo recombineering of bacteriophage λ by PCR fragments and single-strand oligonucleotides. Virology 319:185-189.
references.citation: Russell, C.B. , Thaler, D.S. , and Dahlquist, F.W. 1989. Chromosomal transformation of Escherichia coli recD strains with linearized plasmids. J. Bacteriol. 171:2609-2613.
references.citation: Rybalchenko, N. , Golub, E.I. , Bi, B. , and Radding C.M. 2004. Strand invasion promoted by recombination protein β of coliphage λ. Proc. Natl. Acad. Sci. U.S.A. 101:17056-17060.
references.citation: Sawitzke, J.A. , Thomason, L.C. , Costantino, N. , Bubunenko, M. , Datta, S. , and Court, D.L. 2007. Recombineering: In vivo genetic engineering in E. coli, S. enterica, and beyond. Methods Enzymol. 421:171-199.
references.citation: Sawitzke, J.A. , Costantino, N. , Li, X.T. , Thomason, L.C. , Bubunenko, M. , Court, C. , and Court, D.L. 2011. Probing cellular processes with oligo-mediated recombination and using the knowledge gained to optimize recombineering. J. Mol. Biol. 18:45-59.
references.citation: Schofield, M.J. and Hsieh, P. 2003. DNA mismatch repair: Molecular mechanisms and biological function. Annu. Rev. Microbiol. 57:579-608.
references.citation: Sergueev, K. , Yu, D. , Austin, S. , and Court, D. 2001. Cell toxicity caused by products of the PL operon of bacteriophage lambda. Gene 272:227-235.
references.citation: Sharan, S. , Thomason, L.C. , Kuznetsov S.G. , and Court, DL. 2009. Recombineering: A homologous recombination-based method of genetic engineering. Nat. Protoc. 4:206-223.
references.citation: Stemmer, W.P. , Crameri, A. , Ha, K.D. , Brennan, T.M. , and Heyneker, H.L. 1995. Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. Gene 164:49-53.
references.citation: Swaminathan, S. and Sharan, S.K. 2004. Bacterial artificial chromosome engineering. Methods Mol. Biol. 256:89-106.
references.citation: Swaminathan, S. , Ellis, H.M. , Waters, L.S. , Yu, D. , Lee, E-C. , Court, D.L. , and Sharan, S.K. 2001. Rapid engineering of bacterial artificial chromosomes using oligonucleotides. Genesis 29:14-21.
references.citation: Thomason, L.C. , Myers, R.S. , Oppenheim, A. , Costantino, N. , Sawitzke, J.A. , Datta, S. , Bubunenko, M. , and Court, D.L. 2005. Recombineering in prokaryotes. In Phages: Their Role in Bacterial Pathogenisis and Biotechnology ( M.K. Waldor , D.I. Friedman , and S.L. Adhya , eds.) pp. 383-399. ASM Press, Herndon, Virginia.
references.citation: Thomason, L.C. , Costantino, N. , Shaw, D.V. , and Court, D.L. 2007. Multicopy plasmid modification with phage λ Red recombineering. Plasmid 58:148-158.
references.citation: Yu, D. , Ellis, H.M. , Lee, E.C. , Jenkins, N.A. , Copeland, N.G. , and Court, D.L. 2000. An efficient recombination system for chromosome engineering in Escherichia coli . Proc. Natl. Acad. Sci. U.S.A. 97:5978-5983.
references.citation: Yu, D. , Sawitzke, J.A. , Ellis, H. , and Court, D.L. 2003. Recombineering with overlapping single-stranded DNA oligonucleotides: Testing a recombination intermediate. Proc. Natl. Acad. Sci. U.S.A. 100:7207-7212.
references.citation: Warming, S. , Costantino, N. , Court, D.L. , Jenkins, N.A. , and Copeland, N.G. 2005. Simple and highly efficient BAC recombineering using galK selection. Nucleic Acids Res. 33:e36.
references.citation: Wong, Q.N. , Ng, V.C. , Lin, M.C. , Kung, H.F. , Chan, D. , and Huang, J.D. 2005. Efficient and seamless DNA recombineering using a thymidylate synthase A selection system in Escherichia coli . Nucleic Acids Res. 33:e59.
references.citation: Zhang, Y. , Buchholz, F. , Muyrers, J.P.P. , and Stewart, A.F. 1998. A new logic for DNA engineering using recombination in Escherichia coli . Nat. Genet. 20:123-128.
references.citation: Zhang, Y. , Muyrers, J.P. , Testa, G. , and Stewart, A.F. 2000. DNA cloning by homologous recombination in Escherichia coli . Nat. Biotechnol. 12:1314-1317.