In Vitro Mutagenesis Protocols pp 421-430

Part of the Methods in Molecular Biology book series (MIMB, volume 634)

En Passant Mutagenesis: A Two Step Markerless Red Recombination System

  • B. Karsten Tischer
  • Gregory A. Smith
  • Nikolaus Osterrieder


Bacterial artificial chromosomes are used to maintain and modify large sequences of different origins in Escherichia coli. In addition to RecA-based shuttle mutagenesis, Red recombination is commonly used for sequence modification. Since foreign sequences, such as antibiotic resistance genes as well as frt- or loxP-sites are often unwanted in mutant BAC clones, we developed a Red-based technique that allows for the scarless generation of point mutations, deletions, and insertion of smaller and larger sequences. The method employs a sequence duplication that is inserted into the target sequence in the first recombination step and the excision of the selection marker by in vivo I-SceI cleavage and the second Red recombination. To allow for convenient and highly efficient mutagenesis without the use of additional plasmids, the E. coli strain GS1783 with a chromosomal encoded inducible Red- and I-SceI-expression was created.

Key words

Red recombination BAC Markerless En passant mutagenesis I-SceI 


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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • B. Karsten Tischer
    • 2
  • Gregory A. Smith
    • 1
  • Nikolaus Osterrieder
    • 2
    • 3
  1. 1.Department of Microbiology-ImmunologyNorthwestern UniversityChicagoUSA
  2. 2.Institut für Virologie, Freie Universität BerlinBerlinGermany
  3. 3.Department of Microbiology and ImmunologyCornell UniversityIthacaUSA

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