Rapid Construction of a Whole-genome Transposon Insertion Collection for Shewanella oneidensis by Knockout Sudoku

Abstract

Whole-genome knockout collections are invaluable for connecting gene sequence to function, yet traditionally they have needed an extraordinary technical effort to construct. Knockout Sudoku is a new method for directing the construction and purification of a curated whole-genome collection of singlegene 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 identities of the mutants in 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 3,667 genes. High-throughput kinetic measurements on this collection provide a comprehensive view of multiple extracellular electron transfer pathways operating in parallel.