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.
© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.