ArticleBMC biotechnology2014
A versatile and highly efficient method for scarless genome editing in Escherichia coli and Salmonella enterica.
Article in BMC biotechnology, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 45 papers.
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Who cites it
45 citing papers in PubMed.
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- Metabolic control of porin permeability influences antibiotic resistance in Escherichia coli.Nature microbiology · 2025Article
- Bacterial chromosome conformation and cell-free gene expression in synthetic 2D compartments.Nature communications · 2025Article
- Bioengineered gut bacterium synthesizing levodopa alleviates motor deficits in models of Parkinson's disease.Cell host & microbe · 2025Article
- Selective silencing of antibiotic-tethered ribosomes as a resistance mechanism against aminoglycosides.Nature communications · 2025Article
- Engineering a Robust Escherichia coli W Platform for Scalable Production of Flavonoid-O-Glucosides.Microbial biotechnology · 2025Article
- Mutations Elevate an Underground Pathway to a Physiologically Relevant Protopathway.Molecular biology and evolution · 2025Article
- Article
- A single rare σ70 variant establishes a unique gene expression pattern in the E. coli pathobiont LF82.Nucleic acids research · 2024Article
- Toxin-based screening of C-terminal tags inbioRxiv : the preprint server for biology · 2024Article
- Cobalt complexes modulate plasmid conjugation in Escherichia coli and Klebsiella pneumoniae.Scientific reports · 2024Article
- CRISPR-Cas9-assisted genome editing inbioRxiv : the preprint server for biology · 2024Article
- Methionyl-tRNA synthetase synthetic and proofreading activities are determinants of antibiotic persistence.Frontiers in microbiology · 2024Article
- Polyamines and linear DNA mediate bacterial threat assessment of bacteriophage infection.Proceedings of the National Academy of Sciences of the United States of America · 2023Article
- Interplays of mutations inFrontiers in cellular and infection microbiology · 2023Article
- System metabolic engineering ofFrontiers in bioengineering and biotechnology · 2023Article
- Engineering a suite of E. coli strains for enhanced expression of bacterial polysaccharides and glycoconjugate vaccines.Microbial cell factories · 2022Article
- Absence, loss-of-function, or inhibition of Escherichia coli AcrB does not increase expression of other efflux pump genes supporting the discovery of AcrB inhibitors as antibiotic adjuvants.The Journal of antimicrobial chemotherapy · 2022Article
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Authors and funding
5 authors.
Funding
Abstract
backgroundRecently developed methods for genome editing in bacteria take advantage of the introduction of double-strand breaks by I-SceI in a mutation cassette to select for cells in which homologous recombination has healed the break and introduced a desired mutation. This elegantly designed method did not work well in our hands for most genes.
resultsWe corrected a mutation in the gene encoding I-SceI that compromised the function of a previously used Red helper plasmid. Further, we found that transcription extending into the mutation cassette interferes with cleavage by I-SceI. Addition of two transcription terminators upstream of the cleavage site dramatically increases the efficiency of genome editing. We also developed an improved method for modification of essential genes. Inclusion of a segment of the essential gene consisting of synonymous codons restores an open reading frame when the mutation cassette is integrated into the genome and decreases the frequency of recombination events that fail to incorporate the desired mutation. The optimized protocol takes only 5 days and has been 100% successful for over 100 genomic modifications in our hands.
conclusionsThe method we describe here is reliable and versatile, enabling various types of genome editing in Escherichia coli and Salmonella enterica by straightforward modifications of the mutation cassette. We provide detailed descriptions of the methods as well as designs for insertions, deletions, and introduction of point mutations.
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