This is a paper website for our manuscript. It is a convenient wrapper for the github code repo, which contains html versions of all code notebooks. Our goal is to help others use ORBIT, so we also link to several resources that may be of interest. We welcome any feedback - Thank you!

-Scott Saunders, Distinguished Fellow, UTSW Medical Center

bioRxiv preprint   |   github repository   | Saunders lab ORBIT help page   |   Targeting oligo design app


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 significantly 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.

Code notebooks

Supplemental figures

Other resources

Addgene plasmids

SRA raw sequencing data

Experiment Library Name Run ID and Link Size
galK barcodes galK_BC16N SRR25076308 123 Mb
IDT oPool - left junction oPool_J1 SRR25076307 8.3 Mb
Twist short TF deletions - left junction short_TF_J1 SRR25076303 25 Mb
Twist long TF deletions - left junction long_TF_J1 SRR25076301 16 Mb
Twist small RNA deletions - left junction small_RNA_J1 SRR25076299 18 Mb
IDT oPool - right junction oPool_J2 SRR25076304 15 Mb
Twist short TF deletions - right junction short_TF_J2 SRR25076302 43 Mb
Twist long TF deletions - right junction long_TF_J2 SRR25076300 39 Mb
Twist small RNA deletions - right junction small_RNA_J2 SRR25076298 30 Mb
Twist short TF oligo abundance short_TF_oligos SRR25076297 45 Mb
Twist long TF oligo abundance long_TF_oligos SRR25076306 27 Mb
Twist small RNA oligo abundance small_RNA_oligos SRR25076305 42 Mb