An improved recombineering approach by adding RecA to λ red recombination

Research output: Contribution to journalResearch articleContributedpeer-review

Contributors

  • Junping Wang - (Author)
  • Mihail Sarov - , TUD Dresden University of Technology (Author)
  • Jeanette Rientjes - (Author)
  • Jun Fu - (Author)
  • Heike Hollak - (Author)
  • Harald Kranz - (Author)
  • Wei Xie - (Author)
  • A. Francis Stewart - , Chair of Applied Genomics (Author)
  • Youming Zhang - (Author)

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 Redγ, 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 the stability of cloned DNAs. However, loss of RecA also impairs general cellular integrity. Here we report that transient RecA co-expression enhances the total number 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 pSC101 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.

Details

Original languageEnglish
Pages (from-to)43-53
Number of pages11
JournalMolecular Biotechnology
Volume32
Issue number1
Publication statusPublished - Jan 2006
Peer-reviewedYes

External IDs

PubMed 16382181
ORCID /0000-0002-4754-1707/work/142248114

Keywords

Keywords

  • BAC, Counter selection, recA, Recombineering, Red/ET, λ red