PT  - JOURNAL ARTICLE
AU  - Michael Baym
AU  - Lev Shaket
AU  - Isao A. Anzai
AU  - Oluwakemi Adesina
AU  - Buz Barstow
TI  - Rapid Construction of a Whole-genome Transposon Insertion Collection for <em>Shewanella oneidensis</em> by Knockout Sudoku
AID  - 10.1101/044768
DP  - 2016 Jan 01
TA  - bioRxiv
PG  - 044768
4099  - http://biorxiv.org/content/early/2016/03/21/044768.short
4100  - http://biorxiv.org/content/early/2016/03/21/044768.full
AB  - Whole-genome knockout collections are an invaluable resource for connecting gene sequence to function, yet traditionally, their construction has been an extraordinary technical endeavor. Here, we describe Knockout Sudoku: a new method for directing the construction and purification of a curated whole-genome collection of single-gene disruption mutants generated by transposon mutagenesis. Using a simple combinatorial pooling scheme, a highly oversampled collection of transposon mutants can be condensed into a next-generation sequencing library in a single day. The sequences of the collection are then solved by a predictive algorithm based on Bayesian inference, allowing for rapid curation and validation. Starting from a progenitor collection of 39,918 transposon mutants, we compiled a quality-controlled knockout collection of the electroactive microbe Shewanella oneidensis MR–1 containing representatives for 3,282 genes. High-throughput kinetic measurements on this collection provide a comprehensive view of multiple extracellular electron transfer pathways operating in parallel.