A new recombineering system for precise genome-editing in Shewanella oneidensis strain MR-1 using single-stranded oligonucleotides
Abstract
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. However, 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. Here, 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 . oneidensismore »
- Publication Date:
- Research Org.:
- Univ. of Colorado, Boulder, CO (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC), Biological and Environmental Research (BER); National Institutes of Health (NIH); National Cancer Institute (NCI)
- OSTI Identifier:
- 1619566
- Alternate Identifier(s):
- OSTI ID: 1547416
- Grant/Contract Number:
- SC0018368
- Resource Type:
- Published Article
- Journal Name:
- Scientific Reports
- Additional Journal Information:
- Journal Name: Scientific Reports Journal Volume: 9 Journal Issue: 1; Journal ID: ISSN 2045-2322
- Publisher:
- Nature Publishing Group
- Country of Publication:
- United Kingdom
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES
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 Kingdom: 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 Kingdom. https://doi.org/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 Kingdom. https://doi.org/10.1038/s41598-018-37025-4.
@article{osti_1619566,
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 = {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. However, 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. Here, 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 other environmental bacteria.},
doi = {10.1038/s41598-018-37025-4},
journal = {Scientific Reports},
number = 1,
volume = 9,
place = {United Kingdom},
year = {Thu Jan 10 00:00:00 EST 2019},
month = {Thu Jan 10 00:00:00 EST 2019}
}
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