Dataset Information

Duplication-Insertion Recombineering: a fast and scar-free method for efficient transfer of multiple mutations in bacteria.

ABSTRACT: We have developed a new ? 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 ? 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.

SUBMITTER: Nasvall J

PROVIDER: S-EPMC5389514 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Duplication-Insertion Recombineering: a fast and scar-free method for efficient transfer of multiple mutations in bacteria.

Näsvall Joakim J Knöppel Anna A Andersson Dan I DI

Nucleic acids research 20170301 5

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 eas ...[more]

PMID: 27899661

Similar Datasets

Project description:BackgroundExpression and purification of correctly folded proteins typically require screening of different parameters such as protein variants, solubility enhancing tags or expression hosts. Parallel vector series that cover all variations are available, but not without compromise. We have established a fast, efficient and absolutely background free cloning approach that can be applied to any selected vector.ResultsHere we describe a method to tailor selected expression vectors for parallel Sequence and Ligation Independent Cloning. SLIC cloning enables precise and sequence independent engineering and is based on joining vector and insert with 15-25 bp homologies on both DNA ends by homologous recombination. We modified expression vectors based on pET, pFastBac and pTT backbones for parallel PCR-based cloning and screening in E.coli, insect cells and HEK293E cells, respectively. We introduced the toxic ccdB gene under control of a strong constitutive promoter for counterselection of insert less vector. In contrast to DpnI treatment commonly used to reduce vector background, ccdB used in our vector series is 100% efficient in killing parental vector carrying cells and reduces vector background to zero. In addition, the 3' end of ccdB functions as a primer binding site common to all vectors. The second shared primer binding site is provided by a HRV 3C protease cleavage site located downstream of purification and solubility enhancing tags for tag removal. We have so far generated more than 30 different parallel expression vectors, and successfully cloned and expressed more than 250 genes with this vector series. There is no size restriction for gene insertion, clone efficiency is > 95% with clone numbers up to 200. The procedure is simple, fast, efficient and cost-effective. All expression vectors showed efficient expression of eGFP and different target proteins requested to be produced and purified at our Core Facility services.ConclusionThis new expression vector series allows efficient and cost-effective parallel cloning and thus screening of different protein constructs, tags and expression hosts.

Dataset Information

Duplication-Insertion Recombineering: a fast and scar-free method for efficient transfer of multiple mutations in bacteria.

Publications

Duplication-Insertion Recombineering: a fast and scar-free method for efficient transfer of multiple mutations in bacteria.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets