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Methods Mol Biol. 2010;634:421-30. doi: 10.1007/978-1-60761-652-8_30.

En passant mutagenesis: a two step markerless red recombination system.

Author information

1
Institut für Virologie, Freie Universität Berlin, Berlin, Germany. k.tischer@fu-berlin.de

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.

PMID:
20677001
DOI:
10.1007/978-1-60761-652-8_30
[Indexed for MEDLINE]

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