ArticleNucleic acids research2017
Duplication-Insertion Recombineering: a fast and scar-free method for efficient transfer of multiple mutations in bacteria.
Article in Nucleic acids research, 2017. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 29 papers.
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29 citing papers in PubMed, 43 citations in OpenAlex.
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3 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
We have developed a new λ Red recombineering methodology for generating transient selection markers that can be used to transfer mutations between bacterial strains of both Escherichia coli and Salmonella enterica. The method is fast, simple and allows for the construction of strains with several mutations without any unwanted sequence changes (scar-free). The method uses λ Red recombineering to generate a marker-held tandem duplication, termed Duplication-Insertion (Dup-In). The Dup-Ins can easily be transferred between strains by generalized transduction and are subsequently rapidly lost by homologous recombination between the two copies of the duplicated sequence, leaving no scar sequence or antibiotic resistance cassette behind. We demonstrate the utility of the method by generating several Dup-Ins in E. coli and S. enterica to transfer genetically linked mutations in both essential and non-essential genes. We have successfully used this methodology to re-construct mutants found after various types of selections, and to introduce foreign genes into the two species. Furthermore, recombineering with two overlapping fragments was as efficient as recombineering with the corresponding single large fragment, allowing more complicated constructions without the need for overlap extension PCR.
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