Unit

UNIT 1.16 Recombineering: Genetic Engineering in Bacteria Using Homologous Recombination

  1. Lynn Thomason,
  2. Donald L. Court,
  3. Mikail Bubunenko,
  4. Nina Costantino,
  5. Helen Wilson,
  6. Simanti Datta,
  7. Amos Oppenheim

Published Online: 1 MAY 2005

DOI: 10.1002/0471142727.mb0116s70

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

How to Cite

Thomason, L., Court, D. L., Bubunenko, M., Costantino, N., Wilson, H., Datta, S. and Oppenheim, A. 2005. Recombineering: Genetic Engineering in Bacteria Using Homologous Recombination. Current Protocols in Molecular Biology. 1:V:1.16.

Author Information

  1. National Cancer Institute at Frederick, Frederick, Maryland

Publication History

  1. Published Online: 1 MAY 2005
  2. Published Print: APR 2005

This is not the most recent version of the article. View current version (14 APR 2014)

Abstract

The bacterial chromosome and plasmids can be engineered in vivo by homologous recombination using PCR products and synthetic oligonucleotides as substrates. This is possible because bacteriophage-encoded recombination functions efficiently recombine sequences with homologies as short as 35 to 40 bases. This 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 double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA). Support protocols describe a two-step method of making genetic alterations without leaving any unwanted changes, and a method for retrieving a genetic marker (cloning) from the E. coli chromosome or a coelectroporated DNA fragment and moving it onto a plasmid. A method is also given to screen for unselected mutations. Additional protocols describe removal of defective prophage, methods for recombineering.