En passant mutagenesis: A Two Markerless red recombination system

B. Karsten Tischer*, Gregory A. Smith, Nikolaus Osterrieder

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapter

316 Scopus citations

Abstract

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.

Original languageEnglish (US)
Title of host publicationIn Vitro Mutagenesis Protocols
Subtitle of host publicationThird Edition
EditorsBraman Jeff
Pages421-430
Number of pages10
DOIs
StatePublished - Dec 1 2010

Publication series

NameMethods in Molecular Biology
Volume634
ISSN (Print)1064-3745

Keywords

  • BAC
  • En passant mutagenesis
  • I-SceI
  • Markerless
  • Red recombination

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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