Emulsion culture : A miniaturized library screening system based on micro-droplets in an emulsified medium(METHODS)

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概要

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• 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 50μm 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.

著者

• Ando Daisuke

  Laboratory Of Biodiversity Science School Of Agriculture And Life Sciences University Of Tokyo

• Kojima Takaaki

  Laboratory Of Molecular Biotechnology Graduate School Of Bioagricultural Sciences Nagoya University

• Nakajima Mitsutoshi

  Graduate School Of Life And Environmental Sciences University Of Tsukuba

• Nakajima Mitsutoshi

  Graduate School Of Life And Environmental Sciences Univ. Of Tsukuba

• Kojima Takaaki

  Laboratory Of Chemical Biology Department Of Biological Science Graduate School Of Science Osaka Pre

• Nakano Hideo

  Laboratory Of Molecular Biotechnology Department Of Bioengineering Sciences Graduate School Of Bioag

• Kobayashi Isao

  Food Engineering Division National Food Research Institute Naro

• Kobayashi Isao

  Food Engineering Division National Food Research Institute

• Nagao Nobuhito

  Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya Universit

• Ojima Teruyo

  Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya Universit

• Kawarasaki Yasuaki

  Biomolecular Engineering Laboratory, Graduate School of Nutritional and Environmental Sciences, Univ

• Ojima Teruyo

  Laboratory Of Molecular Biotechnology Graduate School Of Bioagricultural Sciences Nagoya University

• Kawarasaki Yasuaki

  Biomolecular Engineering Lab. School Of Food And Nutritional Sciences University Of Shizuoka:graduat

• Nagao Nobuhito

  Laboratory Of Molecular Biotechnology Graduate School Of Bioagricultural Sciences Nagoya University

• Ando Daisuke

  Laboratory Of Molecular Biotechnology Graduate School Of Bioagricultural Sciences Nagoya University

• Kawarasaki Yasuaki

  Biomolecular Engineering Lab, Graduate School of Integrated Pharmaceutical and Nutritional Sciences, University of Shizuoka:School of Food and Nutritional Sciences, University of Shizuoka

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