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

BioRxiv : the Preprint Server for Biology
Michael BaymBuz Barstow

Abstract

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.

Related Concepts

Microorganism
Biochemical Pathway
Extracellular
Genes
Electron Transport
Mutant Proteins
Whole Genome Amplification
Massively-Parallel Sequencing
Specimen Collection
Electron Microscopy

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