title: Genome-Wide Tuning of Protein Expression Levels to Rapidly Engineer Microbial Traits.
abstract: The reliable engineering of biological systems requires quantitative mapping of predictable and context-independent expression over a broad range of protein expression levels. However, current techniques for modifying expression levels are cumbersome and are not amenable to high-throughput approaches. Here we present major improvements to current techniques through the design and construction of E. coli genome-wide libraries using synthetic DNA cassettes that can tune expression over a ∼10(4) range. The cassettes also contain molecular barcodes that are optimized for next-generation sequencing, enabling rapid and quantitative tracking of alleles that have the highest fitness advantage. We show these libraries can be used to determine which genes and expression levels confer greater fitness to E. coli under different growth conditions.
authors.affiliation: Department of Chemical and Biological Engineering, University of Colorado Boulder , Boulder, Colorado 80309, United States.
authors.forename: Emily F
authors.initials: EF
authors.lastname: Freed
authors.affiliation: Department of Chemical and Biological Engineering, University of Colorado Boulder , Boulder, Colorado 80309, United States.
authors.forename: James D
authors.initials: JD
authors.lastname: Winkler
authors.affiliation: Department of Chemical and Biological Engineering, University of Colorado Boulder , Boulder, Colorado 80309, United States.
authors.forename: Sophie J
authors.initials: SJ
authors.lastname: Weiss
authors.affiliation: Department of Chemical and Biological Engineering, University of Colorado Boulder , Boulder, Colorado 80309, United States.
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Garst
authors.affiliation: Lawrence Berkeley National Laboratory , Physical Biosciences Division, Berkeley, California 94720, United States.|Department of Bioengineering, University of California, Berkeley , Berkeley, California 94720, United States.
authors.forename: Vivek K
authors.initials: VK
authors.lastname: Mutalik
authors.affiliation: Lawrence Berkeley National Laboratory , Physical Biosciences Division, Berkeley, California 94720, United States.|Department of Bioengineering, University of California, Berkeley , Berkeley, California 94720, United States.
authors.forename: Adam P
authors.initials: AP
authors.lastname: Arkin
authors.affiliation: Departments of Pediatrics and Computer Science & Engineering, University of California San Diego , La Jolla, California 92093, United States.
authors.forename: Rob
authors.initials: R
authors.lastname: Knight
authors.affiliation: Department of Chemical and Biological Engineering, University of Colorado Boulder , Boulder, Colorado 80309, United States.
authors.forename: Ryan T
authors.initials: RT
authors.lastname: Gill
mesh_terms: D004926:Escherichia coli / Q000235:genetics*; D005818:Genetic Engineering; D016680:Genome, Bacterial; D058615:Synthetic Biology
keywords: Illumina sequencing; directed evolution; genome-wide expression library; genotype-phenotype mapping; recombineering