An ultra-dense library resource for rapid deconvolution of mutations that cause phenotypes in Escherichia coli
journal contributionposted on 10.02.2016, 09:44 by R. B. Nehring, F. Gu, H. Y. Lin, J. L. Gibson, M. J. Blythe, R. Wilson, M. A. Bravo Núñez, P. J. Hastings, Edward John Louis, R. L. Frisch, J. C. Hu, S. M. Rosenberg
With the wide availability of whole-genome sequencing (WGS), genetic mapping has become the rate-limiting step, inhibiting unbiased forward genetics in even the most tractable model organisms. We introduce a rapid deconvolution resource and method for untagged causative mutations after mutagenesis, screens, and WGS in Escherichia coli. We created Deconvoluter-ordered libraries with selectable insertions every 50 kb in the E. coli genome. The Deconvoluter method uses these for replacement of untagged mutations in the genome using a phage-P1-based gene-replacement strategy. We validate the Deconvoluter resource by deconvolution of 17 of 17 phenotype-altering mutations from a screen of N-ethyl-N-nitrosourea-induced mutants. The Deconvoluter resource permits rapid unbiased screens and gene/function identification and will enable exploration of functions of essential genes and undiscovered genes/sites/alleles not represented in existing deletion collections. This resource for unbiased forward-genetic screens with mapping-by-sequencing ('forward genomics') demonstrates a strategy that could similarly enable rapid screens in many other microbes.