En Passant Mutagenesis: A Two Step Markerless Red Recombination System
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 wordsRed recombination BAC Markerless En passant mutagenesis I-SceI
The authors thank Jens von Einem, Benedikt B. Kaufer, and Felix Wussow for their help in optimizing the presented method. We gratfully recognize Jenifer Klabis for her technical contributions to the isolation of the GS1783 E. coli strain.
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