Recombineering: Genetic Engineering in Bacteria Using Homologous Recombination

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. Curr. Protoc. Mol. Biol. 106:1.16.1‐1.16.39. © 2014 by John Wiley & Sons, Inc.

[1]  Xin-tian Li,et al.  Positive and negative selection using the tetA-sacB cassette: recombineering and P1 transduction in Escherichia coli , 2013, Nucleic acids research.

[2]  Feng Zhang,et al.  CRISPR-assisted editing of bacterial genomes , 2013, Nature Biotechnology.

[3]  Rolf Müller,et al.  Full-length RecE enhances linear-linear homologous recombination and facilitates direct cloning for bioprospecting , 2012, Nature Biotechnology.

[4]  K. Murphy,et al.  Phage recombinases and their applications. , 2012, Advances in virus research.

[5]  A. Hyman,et al.  High-efficiency counterselection recombineering for site-directed mutagenesis in bacterial artificial chromosomes , 2011, Nature Methods.

[6]  N. Costantino,et al.  Probing cellular processes with oligo-mediated recombination and using the knowledge gained to optimize recombineering. , 2011, Journal of molecular biology.

[7]  G. Church,et al.  Lambda Red Recombineering in Escherichia coli Occurs Through a Fully Single-Stranded Intermediate , 2010, Genetics.

[8]  A. Stewart,et al.  Single-stranded heteroduplex intermediates in λ Red homologous recombination , 2010, BMC Molecular Biology.

[9]  D. Court,et al.  Recombineering: a homologous recombination-based method of genetic engineering , 2009, Nature Protocols.

[10]  Xiaomei Zhou,et al.  Identification and analysis of recombineering functions from Gram-negative and Gram-positive bacteria and their phages , 2008, Proceedings of the National Academy of Sciences.

[11]  J. Devito.  Recombineering with tolC as a Selectable/Counter-selectable Marker: remodeling the rRNA Operons of Escherichia coli , 2007, Nucleic acids research.

[12]  N. Costantino,et al.  Multicopy Plasmid Modification with Phage λ Red Recombineering , 2007 .

[13]  D. Court,et al.  Essentiality of Ribosomal and Transcription Antitermination Proteins Analyzed by Systematic Gene Replacement in Escherichia coli , 2007, Journal of bacteriology.

[14]  N. Costantino,et al.  Recombineering: in vivo genetic engineering in E. coli, S. enterica, and beyond. , 2007, Methods in enzymology.

[15]  N. Costantino,et al.  A set of recombineering plasmids for gram-negative bacteria. , 2006, Gene.

[16]  Jian-Dong Huang,et al.  Efficient and seamless DNA recombineering using a thymidylate synthase A selection system in Escherichia coli , 2005, Nucleic acids research.

[17]  Nancy A. Jenkins,et al.  Simple and highly efficient BAC recombineering using galK selection , 2005, Nucleic acids research.

[18]  N. Costantino,et al.  Recombineering in Prokaryotes , 2005 .

[19]  E. Golub,et al.  Strand invasion promoted by recombination protein β of coliphage λ , 2004 .

[20]  D. Court,et al.  In vivo recombineering of bacteriophage λ by PCR fragments and single-strand oligonucleotides , 2004 .

[21]  S. Sharan,et al.  Bacterial artificial chromosome engineering. , 2004, Methods in molecular biology.

[22]  K. Murphy,et al.  Lambda Red-mediated recombinogenic engineering of enterohemorrhagic and enteropathogenic E. coli , 2003, BMC Molecular Biology.

[23]  N. Costantino,et al.  Enhanced levels of λ Red-mediated recombinants in mismatch repair mutants , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[24]  M. Schofield,et al.  DNA mismatch repair: molecular mechanisms and biological function. , 2003, Annual review of microbiology.

[25]  D. Court,et al.  Mini-λ: a tractable system for chromosome and BAC engineering , 2003 .

[26]  Daiguan Yu,et al.  Recombineering with overlapping single-stranded DNA oligonucleotides: Testing a recombination intermediate , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[27]  N. Costantino,et al.  Enhanced levels of lambda Red-mediated recombinants in mismatch repair mutants. , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[28]  D. Court,et al.  Mini-lambda: a tractable system for chromosome and BAC engineering. , 2003, Gene.

[29]  D. Court,et al.  Genetic engineering using homologous recombination. , 2002, Annual review of genetics.

[30]  Nancy A. Jenkins,et al.  Recombineering: a powerful new tool for mouse functional genomics , 2001, Nature Reviews Genetics.

[31]  D. Court,et al.  Cell toxicity caused by products of the p(L) operon of bacteriophage lambda. , 2001, Gene.

[32]  D. Court,et al.  High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[33]  D. Court,et al.  A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA. , 2001, Genomics.

[34]  D. Court,et al.  Rapid engineering of bacterial artificial chromosomes using oligonucleotides , 2000, Genesis.

[35]  Giuseppe Testa,et al.  DNA cloning by homologous recombination in Escherichia coli , 2000, Nature Biotechnology.

[36]  B. Wanner,et al.  One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[37]  D. Court,et al.  An efficient recombination system for chromosome engineering in Escherichia coli. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[38]  K. Murphy,et al.  PCR-mediated gene replacement in Escherichia coli. , 2000, Gene.

[39]  A. Stewart,et al.  Rapid modification of bacterial artificial chromosomes by ET-recombination. , 1999, Nucleic acids research.

[40]  Frank Buchholz,et al.  A new logic for DNA engineering using recombination in Escherichia coli , 1998, Nature Genetics.

[41]  R. Kolodner,et al.  DNA Strand Invasion Promoted by Escherichia coli RecT Protein* , 1998, The Journal of Biological Chemistry.

[42]  W. Stemmer,et al.  Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. , 1995, Gene.

[43]  B. Dixon.  Taking Aim at Sexually Transmitted Diseases , 1994, Bio/Technology.

[44]  R. Kolodner,et al.  Identification and Characterization of the Escherichia coli RecT Protein, a Protein Encoded by the recE Region That Promotes Renaturation of Homologous Single-Stranded DNA , 1993, Journal of bacteriology.

[45]  W. Thilly,et al.  Mismatch amplification mutation assay (MAMA): application to the c-H-ras gene. , 1992, PCR methods and applications.

[46]  I. Blomfield,et al.  Allelic exchange in Escherichia coli using the Bacillus subtilis sacB gene and a temperature‐sensitive pSC101 replicon , 1991, Molecular microbiology.

[47]  E. Myers,et al.  Basic local alignment search tool. , 1990, Journal of molecular biology.

[48]  F. Stahl,et al.  Interaction between the sbcC gene of Escherichia coli and the gam gene of phage lambda. , 1989, Genetics.

[49]  F. Dahlquist,et al.  Chromosomal transformation of Escherichia coli recD strains with linearized plasmids , 1989, Journal of bacteriology.

[50]  A. Cohen,et al.  Synthesis of linear plasmid multimers in Escherichia coli K-12 , 1986, Journal of bacteriology.

[51]  K. Muniyappa,et al.  The homologous recombination system of phage lambda. Pairing activities of beta protein. , 1986, The Journal of biological chemistry.

[52]  D. le Coq,et al.  Positive selection procedure for entrapment of insertion sequence elements in gram-negative bacteria , 1985, Journal of bacteriology.

[53]  R. Kolodner,et al.  Exonuclease VIII of Escherichia coli. II. Mechanism of action. , 1983, The Journal of biological chemistry.

[54]  L. Enquist,et al.  Experimental Methods for Use with Lambda , 1983 .

[55]  Selection for loss of tetracycline resistance by Escherichia coli. , 1981, Journal of bacteriology.

[56]  B. Ames,et al.  Positive selection for loss of tetracycline resistance , 1980, Journal of bacteriology.

[57]  S. Adhya,et al.  Control of transcription termination. , 1978, Annual review of biochemistry.

[58]  Y. Sakaki,et al.  The gamma protein specified by bacteriophage gamma. Structure and inhibitory activity for the recBC enzyme of Escherichia coli. , 1975, The Journal of biological chemistry.

[59]  B. Ames,et al.  Positive selection of mutants with deletions of the gal-chl region of the Salmonella chromosome as a screening procedure for mutagens that cause deletions , 1975, Journal of bacteriology.

[60]  C. Radding,et al.  The Role of Exonuclease and β Protein of Phage λ in Genetic Recombination: II. SUBSTRATE SPECIFICITY AND THE MODE OF ACTION OF λ EXONUCLEASE , 1971 .