ArticleNucleic acids research2024
ORBIT for E. coli: kilobase-scale oligonucleotide recombineering at high throughput and high efficiency.
Article in Nucleic acids research, 2024. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 14 papers.
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Who cites it
14 citing papers in PubMed.
- Applications of transposon-insertion sequencing for understanding bacterial physiology.Microbial genomics · 2026Review
- Oligonucleotide genetics forbioRxiv : the preprint server for biology · 2026Article
- Systematic CRISPRi screening reveals genetic modulators ofbioRxiv : the preprint server for biology · 2026Article
- Advances and Challenges in Mycobacterial Genetic Engineering: Techniques for Knockout, Knockdown and Overexpression.Journal of microbiology and biotechnology · 2025Review
- Recent advances in metabolic engineering of Escherichia coli for riboflavin biosynthesis.World journal of microbiology & biotechnology · 2025Review
- EASY-edit: a toolbox for high-throughput single-step custom genetic editing in bacteria.Nucleic acids research · 2025Article
- The Environment-Dependent Regulatory Landscape of thebioRxiv : the preprint server for biology · 2025Article
- Article
- GoldenBraid2.0Synthetic biology (Oxford, England) · 2025Article
- Deciphering regulatory architectures of bacterial promoters from synthetic expression patterns.PLoS computational biology · 2024Article
- Deciphering regulatory architectures from synthetic single-cell expression patterns.bioRxiv : the preprint server for biology · 2024Article
- Article
- Genetically dissecting the electron transport chain of a soil bacterium reveals a generalizable mechanism for biological phenazine-1-carboxylic acid oxidation.PLoS genetics · 2024Article
- Genetically dissecting the electron transport chain of a soil bacterium reveals a generalizable mechanism for biological phenazine-1-carboxylic acid oxidation.bioRxiv : the preprint server for biology · 2023Article
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2 authors.
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Abstract
Microbiology and synthetic biology depend on reverse genetic approaches to manipulate bacterial genomes; however, existing methods require molecular biology to generate genomic homology, suffer from low efficiency, and are not easily scaled to high throughput. To overcome these limitations, we developed a system for creating kilobase-scale genomic modifications that uses DNA oligonucleotides to direct the integration of a non-replicating plasmid. This method, Oligonucleotide Recombineering followed by Bxb-1 Integrase Targeting (ORBIT) was pioneered in Mycobacteria, and here we adapt and expand it for Escherichia coli. Our redesigned plasmid toolkit for oligonucleotide recombineering achieved significantly higher efficiency than λ Red double-stranded DNA recombineering and enabled precise, stable knockouts (≤134 kb) and integrations (≤11 kb) of various sizes. Additionally, we constructed multi-mutants in a single transformation, using orthogonal attachment sites. At high throughput, we used pools of targeting oligonucleotides to knock out nearly all known transcription factor and small RNA genes, yielding accurate, genome-wide, single mutant libraries. By counting genomic barcodes, we also show ORBIT libraries can scale to thousands of unique members (>30k). This work demonstrates that ORBIT for E. coli is a flexible reverse genetic system that facilitates rapid construction of complex strains and readily scales to create sophisticated mutant libraries.
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