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.
Figure 1. ORBIT overview and proof of principle. (No computational content)
Figure 6. Markerless and scarless ORBIT strategies. (No computational content)
Figure 8. High throughput knockout libraries for transcription factor and small RNA genes.
Figure S1. Target oligo design principles and web app. (No computational content)
Figure S3. Troubleshooting attB only colonies. (No computational content)
Figure S4. Single gene deletion accuracy and stability. (No computational content)
Figure S5. Helper plasmid efficiency and off target SNP mutation rate.
Figure S6. Variable deletion size phenotypic accuracy. (No computational content)
Figure S7. Sanger sequence confirmation of galK deletions of various sizes. (No computational content)
Figure S8. Sanger sequence confirmation of hisA, metA and leuD deletions of various sizes. (No computational content)
Figure S9. Phenotypic accuracy of orthogonal and untargeted double mutants. (No computational content)
[Figure S10. Orthogonal att sites and triple mutant construction.]
Figure S11. oPool library - Upstream and downstream reads for target loci.
Figure S12. Low abundance (Twist Biosciences) targeting oligo library processing. (No computational content)
Figure S13. Short transcription factor library - Upstream and downstream reads for target loci.
Figure S15. Small RNA deletion library - Upstream and downstream reads for target loci.
Figure S17. Correlations of oligo parameters and observed reads per target (perfect reads).
| 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 |