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Molecular Biotechnology

, Volume 32, Issue 1, pp 43–53 | Cite as

An improved recombineering approach by adding RecA to λ Red recombination

  • Junping Wang
  • Mihail Sarov
  • Jeanette Rientjes
  • Jun Hu
  • Heike Hollak
  • Harald Kranz
  • Yei Xie
  • A. Francis Stewart
  • Youming ZhangEmail author
Research

Abstract

Recombineering is the use of homologous recombination in Escherichia coli for DNA engineering. Of several approaches, use of the λ phage Red operon is emerging as the most reliable and flexible. The Red operon includes three components: Redα, a 5′ to 3′ exonuclease, Redβ, an annealing protein, and Redλ, an inhibitor of the major E. coli exonuclease and recombination complex, RecBCD. Most E. coli cloning hosts are recA deficient to eliminate recombination and therefore enhance thestabulity of cloned DNAs. However, loss of RecA also impairs general cellular integrity. Here we report that transient RecA co-expression enhances the total numer of successful recombinations in bacterial artificial chromosomes (BACs), mostly because the E. coli host is more able to survive the stresses of DNA transformation procedures. We combined this practical improvement with the advantages of a temperature-sensitive version of the low copy pSC 101 plasmid to develop a protocol that is convenient and more efficient than any recombineering procedure, for use of either double-or single-stranded DNA, published to date.

Index Entries

Recombineering Red/ET recA λ red counter selection BAC 

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Copyright information

© Humana Press Inc 2006

Authors and Affiliations

  • Junping Wang
    • 1
  • Mihail Sarov
    • 2
  • Jeanette Rientjes
    • 1
  • Jun Hu
    • 1
  • Heike Hollak
    • 1
  • Harald Kranz
    • 1
  • Yei Xie
    • 1
  • A. Francis Stewart
    • 2
  • Youming Zhang
    • 1
    Email author
  1. 1.BiolnnovationsZentrum DresdenGene Bridges GmbHDresdenGermany
  2. 2.Biotec, Genomics, University of Technology DresdenBrolnnovationsZentrum DresdenDresdenGermany

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