RT Journal Article SR Electronic T1 ORBIT for E. coli: Kilobase-scale oligonucleotide recombineering at high throughput and high efficiency JF bioRxiv FD Cold Spring Harbor Laboratory SP 2023.06.28.546561 DO 10.1101/2023.06.28.546561 A1 Saunders, Scott H. A1 Ahmed, Ayesha M. YR 2023 UL http://biorxiv.org/content/early/2023/07/11/2023.06.28.546561.abstract AB 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 applications. 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 E. coli. Our redesigned plasmid toolkit achieved nearly 1000x higher efficiency than λ Red recombination and enabled precise, stable knockouts (<134 kb) and integrations (<11 kb) of various sizes. Additionally, we constructed multi-mutants (double and triple) 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.Competing Interest StatementThe authors have declared no competing interest.