Molecular Biotechnology

, Volume 32, Issue 1, pp 43–53

An improved recombineering approach by adding RecA to λ Red recombination

Authors

  • Junping Wang
    • BiolnnovationsZentrum DresdenGene Bridges GmbH
  • Mihail Sarov
    • Biotec, Genomics, University of Technology DresdenBrolnnovationsZentrum Dresden
  • Jeanette Rientjes
    • BiolnnovationsZentrum DresdenGene Bridges GmbH
  • Jun Hu
    • BiolnnovationsZentrum DresdenGene Bridges GmbH
  • Heike Hollak
    • BiolnnovationsZentrum DresdenGene Bridges GmbH
  • Harald Kranz
    • BiolnnovationsZentrum DresdenGene Bridges GmbH
  • Yei Xie
    • BiolnnovationsZentrum DresdenGene Bridges GmbH
  • A. Francis Stewart
    • Biotec, Genomics, University of Technology DresdenBrolnnovationsZentrum Dresden
    • BiolnnovationsZentrum DresdenGene Bridges GmbH
Research

DOI: 10.1385/MB:32:1:043

Cite this article as:
Wang, J., Sarov, M., Rientjes, J. et al. Mol Biotechnol (2006) 32: 43. doi:10.1385/MB:32:1:043

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

RecombineeringRed/ETrecAλ redcounter selectionBAC
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Copyright information

© Humana Press Inc 2006