{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,4]],"date-time":"2026-09-04T03:35:39Z","timestamp":1788492939664,"version":"build-2803163510"},"reference-count":67,"publisher":"Oxford University Press (OUP)","issue":"8","license":[{"start":{"date-parts":[[2024,4,8]],"date-time":"2024-04-08T00:00:00Z","timestamp":1712534400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2024,5,8]]},"abstract":"<jats:title>Abstract<\/jats:title>\n               <jats:p>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\u00a0coli. Our redesigned plasmid toolkit for oligonucleotide recombineering achieved significantly higher efficiency than \u03bb Red double-stranded DNA recombineering and enabled precise, stable knockouts (\u2264134 kb) and integrations (\u226411 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 (&amp;gt;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.<\/jats:p>","DOI":"10.1093\/nar\/gkae227","type":"journal-article","created":{"date-parts":[[2024,4,8]],"date-time":"2024-04-08T11:03:05Z","timestamp":1712574185000},"page":"e43-e43","source":"Crossref","is-referenced-by-count":14,"title":["ORBIT for <i>E. coli<\/i>: kilobase-scale oligonucleotide recombineering at high throughput and high efficiency"],"prefix":"10.1093","volume":"52","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4224-9106","authenticated-orcid":false,"given":"Scott H","family":"Saunders","sequence":"first","affiliation":[{"name":"Green Center for Systems Biology - Lyda Hill Department of Bioinformatics, University of Texas Southwestern Medical Center , Dallas , TX\u00a0 75320 ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ayesha M","family":"Ahmed","sequence":"additional","affiliation":[{"name":"Green Center for Systems Biology - Lyda Hill Department of Bioinformatics, University of Texas Southwestern Medical Center , Dallas , TX\u00a0 75320 ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2024,4,8]]},"reference":[{"key":"2024112915054218900_B1","doi-asserted-by":"crossref","first-page":"526","DOI":"10.1038\/s41576-020-0244-x","article-title":"A decade of advances in transposon-insertion sequencing","volume":"21","author":"Cain","year":"2020","journal-title":"Nat. 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