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Rapid construction of a whole-genome transposon insertion collection for Shewanella oneidensis by Knockout Sudoku

Author

Listed:
  • Michael Baym

    (Harvard Medical School)

  • Lev Shaket

    (Princeton University)

  • Isao A. Anzai

    (Princeton University)

  • Oluwakemi Adesina

    (Princeton University)

  • Buz Barstow

    (Princeton University)

Abstract

Whole-genome knockout collections are invaluable for connecting gene sequence to function, yet traditionally, their construction has required an extraordinary technical effort. Here we report a method for the construction and purification of a curated whole-genome collection of single-gene transposon disruption mutants termed Knockout Sudoku. Using simple combinatorial pooling, a highly oversampled collection of mutants is condensed into a next-generation sequencing library in a single day, a 30- to 100-fold improvement over prior methods. The identities of the mutants in the collection are then solved by a probabilistic algorithm that uses internal self-consistency within the sequencing data set, followed by rapid algorithmically guided condensation to a minimal representative set of mutants, validation, and curation. Starting from a progenitor collection of 39,918 mutants, we compile a quality-controlled knockout collection of the electroactive microbe Shewanella oneidensis MR-1 containing representatives for 3,667 genes that is functionally validated by high-throughput kinetic measurements of quinone reduction.

Suggested Citation

  • Michael Baym & Lev Shaket & Isao A. Anzai & Oluwakemi Adesina & Buz Barstow, 2016. "Rapid construction of a whole-genome transposon insertion collection for Shewanella oneidensis by Knockout Sudoku," Nature Communications, Nature, vol. 7(1), pages 1-13, December.
    • Handle: RePEc:nat:natcom:v:7:y:2016:i:1:d:10.1038_ncomms13270
    DOI: 10.1038/ncomms13270
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    Cited by:

    1. Alexa M. Schmitz & Brooke Pian & Sean Medin & Matthew C. Reid & Mingming Wu & Esteban Gazel & Buz Barstow, 2021. "Generation of a Gluconobacter oxydans knockout collection for improved extraction of rare earth elements," Nature Communications, Nature, vol. 12(1), pages 1-11, December.

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