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

Corts, Anna D.; Thomason, Lynn C.; Gill, Ryan T.; Gralnick, Jeffrey A.

doi:10.1038/s41598-018-37025-4

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

Journal Article · 09 January 2019 · Scientific Reports

DOI: https://doi.org/10.1038/s41598-018-37025-4 · OSTI ID:1619566

Corts, Anna D.; Thomason, Lynn C.; Gill, Ryan T.;

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.

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Research Organization:: Univ. of Colorado, Boulder, CO (United States)

Sponsoring Organization:: National Cancer Institute; National Institutes of Health (NIH); USDOE; USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)

Grant/Contract Number:: SC0018368

OSTI ID:: 1619566

Journal Information:: Scientific Reports, Journal Name: Scientific Reports Journal Issue: 1 Vol. 9; ISSN 2045-2322

Publisher:: Nature Publishing GroupCopyright Statement

Country of Publication:: United Kingdom

Language:: English

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