title: Emulsion culture: a miniaturized library screening system based on micro-droplets in an emulsified medium.
abstract: A typical library screen in directed evolution primarily requires physical separation of the clones on agar plates followed by detection of clones with improved properties; using this method only limited numbers of clones relative to the number of potential variations can be assessed. In particular, screening for a secretory enzyme is difficult to perform at high clone density, because of diffusion of the signal or unfavorable utilization of the reaction product by neighboring clones. In this study, we have developed a novel method of enrichment culture: "Emulsion Culture", i.e., segregated replication of clones in an emulsified culture medium. Clones expressing enzyme-variants are separately distributed to small (up to 50 μm in diameter), segregated compartments composed of a droplet of medium to form several tens of millions of microcolonies in a milliliter of medium, which allows a miniaturized, in-bulk screening of clones. We applied this culture method to yeast clones expressing secretory beta-galactosidase to analyze the enrichment factor achieved. A high-density screen for a signal peptide sequence that maximizes extracellular production of the enzyme was also performed to demonstrate the practicability of this culture method. In addition, micro-channel emulsification was tested as a method of forming uniformly-sized compartments in the emulsion.
authors.affiliation: Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Japan.
authors.forename: Takaaki
authors.initials: T
authors.lastname: Kojima
authors.forename: Nobuhito
authors.initials: N
authors.lastname: Nagao
authors.forename: Daisuke
authors.initials: D
authors.lastname: Ando
authors.forename: Teruyo
authors.initials: T
authors.lastname: Ojima
authors.forename: Yasuaki
authors.initials: Y
authors.lastname: Kawarasaki
authors.forename: Isao
authors.initials: I
authors.lastname: Kobayashi
authors.forename: Mitsutoshi
authors.initials: M
authors.lastname: Nakajima
authors.forename: Hideo
authors.initials: H
authors.lastname: Nakano
mesh_terms: D000595:Amino Acid Sequence; D019411:Clinical Laboratory Techniques; D004655:Emulsions / Q000737:chemistry; D006801:Humans; D019151:Peptide Library; D021382:Protein Sorting Signals; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D001616:beta-Galactosidase / Q000378:metabolism*
chemical_list: D004655:Emulsions; D019151:Peptide Library; D021382:Protein Sorting Signals; D001616:beta-Galactosidase