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Recombineering in Vibrio natriegens

Henry H. Lee, Nili Ostrov, Michaela A. Gold, George M. Church
doi: https://doi.org/10.1101/130088
Henry H. Lee
1Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
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Nili Ostrov
1Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
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Michaela A. Gold
2Department of Biology, Chemical and Biological Engineering, Tufts University, Medford, Massachusetts, 02155.
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George M. Church
1Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
3Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts 02115, USA.
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Abstract

Here, we show that λ-Red homologs found in the Vibrio-associated SXT mobile element potentiate allelic exchange in V. natriegens by 10,000-fold. Specifically, we show SXT-Beta (s065), SXT-Exo (s066), and λ-Gam proteins are sufficient to enable recombination of single- and double-stranded DNA with episomal and genomic loci. We characterize and optimize episomal oligonucleotide-mediated recombineering and demonstrate recombineering at genomic loci. We further show targeted genomic deletion of the extracellular nuclease gene dns using a double-stranded DNA cassette. Continued development of this recombination technology will advance high-throughput and large-scale genetic engineering efforts to domesticate V. natriegens and to investigate its rapid growth rate.

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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 4.0 International license.
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Posted April 24, 2017.
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Recombineering in Vibrio natriegens
Henry H. Lee, Nili Ostrov, Michaela A. Gold, George M. Church
bioRxiv 130088; doi: https://doi.org/10.1101/130088
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Recombineering in Vibrio natriegens
Henry H. Lee, Nili Ostrov, Michaela A. Gold, George M. Church
bioRxiv 130088; doi: https://doi.org/10.1101/130088

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