Unit

UNIT 1.16 Recombineering: Genetic Engineering in Bacteria Using Homologous Recombination

  1. Lynn C. Thomason1,
  2. James A. Sawitzke2,
  3. Xintian Li2,
  4. Nina Costantino2,
  5. Donald L. Court2

Published Online: 14 APR 2014

DOI: 10.1002/0471142727.mb0116s106

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

How to Cite

Thomason, L. C., Sawitzke, J. A., Li, X., Costantino, N. and Court, D. L. 2014. Recombineering: Genetic Engineering in Bacteria Using Homologous Recombination. Current Protocols in Molecular Biology. 106:V:1.16:1.16.1–1.16.39.

Author Information

  1. 1

    Basic Science Program, GRCBL-Molecular Control & Genetics Section, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc, Frederick, Maryland

  2. 2

    Molecular Control and Genetics Section, Gene Regulation and Chromosome Biology, National Cancer Institute at Frederick, National Institutes of Health, Frederick, Maryland

Publication History

  1. Published Online: 14 APR 2014

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

Keywords:

  • recombineering;
  • bacteria;
  • homologous recombination;
  • bacteriophage λ;
  • λ Red system;
  • RecET;
  • Rac prophage;
  • selection/counter-selection