Skip to main content
bioRxiv
  • Home
  • About
  • Submit
  • ALERTS / RSS
Advanced Search
New Results

ORBIT for E. coli: Kilobase-scale oligonucleotide recombineering at high throughput and high efficiency

View ORCID ProfileScott H. Saunders, Ayesha M. Ahmed
doi: https://doi.org/10.1101/2023.06.28.546561
Scott H. Saunders
1Green Center for Systems Biology - Lyda Hill Department of Bioinformatics, University of Texas Southwestern Medical Center, Dallas, TX 75320
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Scott H. Saunders
  • For correspondence: scott.saunders@utsouthwestern.edu
Ayesha M. Ahmed
1Green Center for Systems Biology - Lyda Hill Department of Bioinformatics, University of Texas Southwestern Medical Center, Dallas, TX 75320
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • Abstract
  • Full Text
  • Info/History
  • Metrics
  • Supplementary material
  • Data/Code
  • Preview PDF
Loading

Abstract

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 Statement

The authors have declared no competing interest.

Footnotes

  • Minor typos, supplemental files, SRA identifiers, and Addgene identifiers.

  • https://github.com/saunders-lab/ecoli_orbit

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC 4.0 International license.
Back to top
PreviousNext
Posted July 11, 2023.
Download PDF

Supplementary Material

Data/Code
Email

Thank you for your interest in spreading the word about bioRxiv.

NOTE: Your email address is requested solely to identify you as the sender of this article.

Enter multiple addresses on separate lines or separate them with commas.
ORBIT for E. coli: Kilobase-scale oligonucleotide recombineering at high throughput and high efficiency
(Your Name) has forwarded a page to you from bioRxiv
(Your Name) thought you would like to see this page from the bioRxiv website.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
ORBIT for E. coli: Kilobase-scale oligonucleotide recombineering at high throughput and high efficiency
Scott H. Saunders, Ayesha M. Ahmed
bioRxiv 2023.06.28.546561; doi: https://doi.org/10.1101/2023.06.28.546561
Reddit logo Twitter logo Facebook logo LinkedIn logo Mendeley logo
Citation Tools
ORBIT for E. coli: Kilobase-scale oligonucleotide recombineering at high throughput and high efficiency
Scott H. Saunders, Ayesha M. Ahmed
bioRxiv 2023.06.28.546561; doi: https://doi.org/10.1101/2023.06.28.546561

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
  • Tweet Widget
  • Facebook Like
  • Google Plus One

Subject Area

  • Microbiology
Subject Areas
All Articles
  • Animal Behavior and Cognition (4667)
  • Biochemistry (10332)
  • Bioengineering (7652)
  • Bioinformatics (26277)
  • Biophysics (13497)
  • Cancer Biology (10663)
  • Cell Biology (15388)
  • Clinical Trials (138)
  • Developmental Biology (8479)
  • Ecology (12799)
  • Epidemiology (2067)
  • Evolutionary Biology (16817)
  • Genetics (11378)
  • Genomics (15451)
  • Immunology (10591)
  • Microbiology (25139)
  • Molecular Biology (10186)
  • Neuroscience (54316)
  • Paleontology (399)
  • Pathology (1663)
  • Pharmacology and Toxicology (2889)
  • Physiology (4331)
  • Plant Biology (9223)
  • Scientific Communication and Education (1585)
  • Synthetic Biology (2551)
  • Systems Biology (6769)
  • Zoology (1459)