Abstract
This chapter describes two related recombineering-based techniques: “Duplication Insertion” (Dup-In) and “Direct- and Inverted Repeat stimulated excision” (DIRex). Dup-In is used for transferring existing mutations between strains, and DIRex for generating almost any type of mutation. Both techniques use intermediate insertions with counter-selectable cassettes, flanked by directly repeated sequences that enable exact and spontaneous excision of the cassettes. These constructs can be transferred to other strains using generalized transductions, and the final intended mutation is obtained following selection for spontaneous loss of the counter-selectable cassette, which leaves only the intended mutation behind in the final strain. The techniques have been used in several strains of Escherichia coli and Salmonella enterica, and should be readily adaptable to other organisms where λ Red recombineering or similar methods are available.
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Näsvall, J. (2022). Dup-In and DIRex: Techniques for Single-Step, Scar-Free Mutagenesis with Marker Recycling. In: Reisch, C.R. (eds) Recombineering. Methods in Molecular Biology, vol 2479. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2233-9_7
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DOI: https://doi.org/10.1007/978-1-0716-2233-9_7
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