ArticlePloS one2017
Direct and Inverted Repeat stimulated excision (DIRex): Simple, single-step, and scar-free mutagenesis of bacterial genes.
Article in PloS one, 2017. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 19 papers.
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19 citing papers in PubMed, 26 citations in OpenAlex.
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- Pulsatile basal gene expression as a fitness determinant in bacteria.Proceedings of the National Academy of Sciences of the United States of America · 2025Article
- Anti-correlation of LacI association and dissociation rates observed in living cells.Nature communications · 2025Article
- Regulatory elements coordinating initiation of chromosome replication to theProceedings of the National Academy of Sciences of the United States of America · 2023Article
- Low levels of tetracyclines select for a mutation that prevents the evolution of high-level resistance to tigecycline.PLoS biology · 2022Article
- Positive Selection during Niche Adaptation Results in Large-Scale and Irreversible Rearrangement of Chromosomal Gene Order in Bacteria.Molecular biology and evolution · 2022Article
- Phenotypic and genetic barriers to establishment of horizontally transferred genes encoding ribosomal protection proteins.The Journal of antimicrobial chemotherapy · 2021Article
- Genetic Architecture and Fitness of Bacterial Interspecies Hybrids.Molecular biology and evolution · 2021Article
- ESI mutagenesis: a one-step method for introducing mutations into bacterial artificial chromosomes.Life science alliance · 2021Article
- Mutational Pathways and Trade-Offs Between HisA and TrpF Functions: Implications for Evolution via Gene Duplication and Divergence.Frontiers in microbiology · 2020Article
- Genetic Engineering by DNA Recombineering.Current protocols in chemical biology · 2019Article
- Structural mechanism of AadA, a dual-specificity aminoglycoside adenylyltransferase fromThe Journal of biological chemistry · 2018Article
- Genetic Adaptation to Growth Under Laboratory Conditions inFrontiers in microbiology · 2018Article
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1 author at 1 institution in 1 country.
Funding
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Abstract
The need for generating precisely designed mutations is common in genetics, biochemistry, and molecular biology. Here, I describe a new λ Red recombineering method (Direct and Inverted Repeat stimulated excision; DIRex) for fast and easy generation of single point mutations, small insertions or replacements as well as deletions of any size, in bacterial genes. The method does not leave any resistance marker or scar sequence and requires only one transformation to generate a semi-stable intermediate insertion mutant. Spontaneous excision of the intermediate efficiently and accurately generates the final mutant. In addition, the intermediate is transferable between strains by generalized transductions, enabling transfer of the mutation into multiple strains without repeating the recombineering step. Existing methods that can be used to accomplish similar results are either (i) more complicated to design, (ii) more limited in what mutation types can be made, or (iii) require expression of extrinsic factors in addition to λ Red. I demonstrate the utility of the method by generating several deletions, small insertions/replacements, and single nucleotide exchanges in Escherichia coli and Salmonella enterica. Furthermore, the design parameters that influence the excision frequency and the success rate of generating desired point mutations have been examined to determine design guidelines for optimal efficiency.
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