Publikationsdatenbank

En passant mutagenesis:
a two step markerless red recombination system (2010)

Art

Zeitschriftenartikel / wissenschaftlicher Beitrag

Autoren

Tischer, B. Karsten (WE 5)

Smith, Gregory A.

Osterrieder, Nikolaus (WE 5)

Quelle

Methods in molecular biology

Bandzählung: 634

Seiten: 421 – 430

ISSN: 1064-3745

Sprache

Englisch

Verweise

URL (Volltext): https://link.springer.com/protocol/10.1007%2F978-1-60761-652-8_30

DOI: 10.1007/978-1-60761-652-8_30

Pubmed: 20677001

Kontakt

Institut für Virologie

Robert-von-Ostertag-Str. 7-13
14163 Berlin
+49 30 838 51833
virologie@vetmed.fu-berlin.de

Abstract / Zusammenfassung

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.