Recombineering in Vibrio natriegens

Here, we show that -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 -Gam proteins are sufficient to enable recombination of single- and double- stranded DNA with epi...

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Bibliographic Details
Published inbioRxiv
Main Authors Lee, Henry H, Ostrov, Nili, Gold, Michaela A, Church, George M
Format Paper
LanguageEnglish
Published Cold Spring Harbor Cold Spring Harbor Laboratory Press 26.04.2017
Subjects
Deoxyribonucleic acid
DNA
Gene deletion
Gene loci
Genetic engineering
Growth rate
Nuclease
Oligonucleotides
Recombination
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Summary:Here, we show that -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 -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.
DOI:10.1101/130088