An ultra-dense library resource for rapid deconvolution of mutations that cause phenotypes in Escherichia coli

作者：Nehring Ralf B; Gu Franklin; Lin Hsin Yu; Gibson Janet L; Blythe Martin J; Wilson Ray; Bravo Nunez Maria Angelica; Hastings P J; Louis Edward J; Frisch Ryan L; Hu James C; Rosenberg Susan M^*

来源：Nucleic Acids Research, 2016, 44(5): e41.

DOI：10.1093/nar/gkv1131

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.

• 出版日期2016-3-18
• 单位河北医科大学