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

Improved bacterial recombineering by parallelized protein discovery

View ORCID ProfileTimothy M. Wannier, View ORCID ProfileAkos Nyerges, Helene M. Kuchwara, Márton Czikkely, Dávid Balogh, View ORCID ProfileGabriel T. Filsinger, Nathaniel C. Borders, View ORCID ProfileChristopher J. Gregg, View ORCID ProfileMarc J. Lajoie, View ORCID ProfileXavier Rios, View ORCID ProfileCsaba Pál, View ORCID ProfileGeorge M. Church
doi: https://doi.org/10.1101/2020.01.14.906594
Timothy M. Wannier
1Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Timothy M. Wannier
  • For correspondence: timothy_wannier@hms.harvard.edu gchurch@genetics.harvard.edu
Akos Nyerges
1Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115
2Synthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre, Szeged, HU-6726, Hungary
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Akos Nyerges
Helene M. Kuchwara
1Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Márton Czikkely
2Synthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre, Szeged, HU-6726, Hungary
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Dávid Balogh
2Synthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre, Szeged, HU-6726, Hungary
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Gabriel T. Filsinger
1Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Gabriel T. Filsinger
Nathaniel C. Borders
1Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Christopher J. Gregg
1Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Christopher J. Gregg
Marc J. Lajoie
1Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Marc J. Lajoie
Xavier Rios
1Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Xavier Rios
Csaba Pál
2Synthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre, Szeged, HU-6726, Hungary
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Csaba Pál
George M. Church
1Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for George M. Church
  • For correspondence: timothy_wannier@hms.harvard.edu gchurch@genetics.harvard.edu
  • Abstract
  • Full Text
  • Info/History
  • Metrics
  • Supplementary material
  • Preview PDF
Loading

Abstract

Exploiting bacteriophage-derived homologous recombination processes has enabled precise, multiplex editing of microbial genomes and the construction of billions of customized genetic variants in a single day. The techniques that enable this, Multiplex Automated Genome Engineering (MAGE) and directed evolution with random genomic mutations (DIvERGE), are however currently limited to a handful of microorganisms for which single-stranded DNA-annealing proteins (SSAPs) that promote efficient recombineering have been identified. Thus, to enable genome-scale engineering in new hosts, highly efficient SSAPs must first be found. Here we introduce a high-throughput method for SSAP discovery that we call Serial Enrichment for Efficient Recombineering (SEER). By performing SEER in E. coli to screen hundreds of putative SSAPs, we identify highly active variants PapRecT and CspRecT. CspRecT increases the efficiency of single-locus editing to as high as 50% and improves multiplex editing by 5 to 10-fold in E. coli, while PapRecT enables efficient recombineering in Pseudomonas aeruginosa, a concerning human pathogen. CspRecT and PapRecT are also active in other, clinically and biotechnologically relevant enterobacteria. We envision that the deployment of SEER in new species will pave the way toward pooled interrogation of genotype-to-phenotype relationships in previously intractable bacteria.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
Back to top
PreviousNext
Posted January 16, 2020.
Download PDF

Supplementary Material

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.
Improved bacterial recombineering by parallelized protein discovery
(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
Improved bacterial recombineering by parallelized protein discovery
Timothy M. Wannier, Akos Nyerges, Helene M. Kuchwara, Márton Czikkely, Dávid Balogh, Gabriel T. Filsinger, Nathaniel C. Borders, Christopher J. Gregg, Marc J. Lajoie, Xavier Rios, Csaba Pál, George M. Church
bioRxiv 2020.01.14.906594; doi: https://doi.org/10.1101/2020.01.14.906594
Reddit logo Twitter logo Facebook logo LinkedIn logo Mendeley logo
Citation Tools
Improved bacterial recombineering by parallelized protein discovery
Timothy M. Wannier, Akos Nyerges, Helene M. Kuchwara, Márton Czikkely, Dávid Balogh, Gabriel T. Filsinger, Nathaniel C. Borders, Christopher J. Gregg, Marc J. Lajoie, Xavier Rios, Csaba Pál, George M. Church
bioRxiv 2020.01.14.906594; doi: https://doi.org/10.1101/2020.01.14.906594

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 (4659)
  • Biochemistry (10313)
  • Bioengineering (7641)
  • Bioinformatics (26246)
  • Biophysics (13481)
  • Cancer Biology (10650)
  • Cell Biology (15366)
  • Clinical Trials (138)
  • Developmental Biology (8468)
  • Ecology (12778)
  • Epidemiology (2067)
  • Evolutionary Biology (16795)
  • Genetics (11373)
  • Genomics (15431)
  • Immunology (10582)
  • Microbiology (25087)
  • Molecular Biology (10172)
  • Neuroscience (54239)
  • Paleontology (398)
  • Pathology (1660)
  • Pharmacology and Toxicology (2884)
  • Physiology (4328)
  • Plant Biology (9214)
  • Scientific Communication and Education (1582)
  • Synthetic Biology (2545)
  • Systems Biology (6762)
  • Zoology (1459)