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Title: A new recombineering system for precise genome-editing in Shewanella oneidensis strain MR-1 using single-stranded oligonucleotides

Abstract

Shewanella oneidensis MR-1 is an invaluable host for the discovery and engineering of pathways important for bioremediation of toxic and radioactive metals and understanding extracellular electron transfer. Yet, genetic manipulation is challenging due to the lack of genetic tools. Previously, the only reliable method used for introducing DNA into Shewanella spp. at high efficiency was bacterial conjugation, enabling transposon mutagenesis and targeted knockouts using suicide vectors for gene disruptions. In this work, we describe development of a robust and simple electroporation method in S. oneidensis that allows an efficiency of ~4.0 x 10 6 transformants/µg DNA. High transformation efficiency is maintained when cells are frozen for long term storage. In addition, we report a new prophage-mediated genome engineering (recombineering) system using a λ Red Beta homolog from Shewanella sp. W3-18-1. By targeting two different chromosomal alleles, we demonstrate its application for precise genome editing using single strand DNA oligonucleotides and show that an efficiency of ~5% recombinants among total cells can be obtained. This is the first effective and simple strategy for recombination with markerless mutations in S. oneidensis. Continued development of this recombinant technology will advance high-throughput and genome modification efforts to engineer and investigate S. oneidensis and othermore » environmental bacteria.« less

Authors:
 [1];  [2];  [3]; ORCiD logo [1]
  1. Univ. of Minnesota-Twin Cities, St. Paul, MN (United States)
  2. Frederick National Lab. for Cancer Research, Frederick, MD (United States)
  3. Univ. of Colorado, Boulder, CO (United States)
Publication Date:
Research Org.:
Univ. of Colorado, Boulder, CO (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23); National Institutes of Health (NIH); National Cancer Institute (NCI)
OSTI Identifier:
1547416
Grant/Contract Number:  
SC0018368
Resource Type:
Accepted Manuscript
Journal Name:
Scientific Reports
Additional Journal Information:
Journal Volume: 9; Journal Issue: 1; Journal ID: ISSN 2045-2322
Publisher:
Nature Publishing Group
Country of Publication:
United States
Language:
English

Citation Formats

Corts, Anna D., Thomason, Lynn C., Gill, Ryan T., and Gralnick, Jeffrey A. A new recombineering system for precise genome-editing in Shewanella oneidensis strain MR-1 using single-stranded oligonucleotides. United States: N. p., 2019. Web. doi:10.1038/s41598-018-37025-4.
Corts, Anna D., Thomason, Lynn C., Gill, Ryan T., & Gralnick, Jeffrey A. A new recombineering system for precise genome-editing in Shewanella oneidensis strain MR-1 using single-stranded oligonucleotides. United States. doi:10.1038/s41598-018-37025-4.
Corts, Anna D., Thomason, Lynn C., Gill, Ryan T., and Gralnick, Jeffrey A. Thu . "A new recombineering system for precise genome-editing in Shewanella oneidensis strain MR-1 using single-stranded oligonucleotides". United States. doi:10.1038/s41598-018-37025-4. https://www.osti.gov/servlets/purl/1547416.
@article{osti_1547416,
title = {A new recombineering system for precise genome-editing in Shewanella oneidensis strain MR-1 using single-stranded oligonucleotides},
author = {Corts, Anna D. and Thomason, Lynn C. and Gill, Ryan T. and Gralnick, Jeffrey A.},
abstractNote = {Shewanella oneidensis MR-1 is an invaluable host for the discovery and engineering of pathways important for bioremediation of toxic and radioactive metals and understanding extracellular electron transfer. Yet, genetic manipulation is challenging due to the lack of genetic tools. Previously, the only reliable method used for introducing DNA into Shewanella spp. at high efficiency was bacterial conjugation, enabling transposon mutagenesis and targeted knockouts using suicide vectors for gene disruptions. In this work, we describe development of a robust and simple electroporation method in S. oneidensis that allows an efficiency of ~4.0 x 106 transformants/µg DNA. High transformation efficiency is maintained when cells are frozen for long term storage. In addition, we report a new prophage-mediated genome engineering (recombineering) system using a λ Red Beta homolog from Shewanella sp. W3-18-1. By targeting two different chromosomal alleles, we demonstrate its application for precise genome editing using single strand DNA oligonucleotides and show that an efficiency of ~5% recombinants among total cells can be obtained. This is the first effective and simple strategy for recombination with markerless mutations in S. oneidensis. Continued development of this recombinant technology will advance high-throughput and genome modification efforts to engineer and investigate S. oneidensis and other environmental bacteria.},
doi = {10.1038/s41598-018-37025-4},
journal = {Scientific Reports},
number = 1,
volume = 9,
place = {United States},
year = {2019},
month = {1}
}

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