Emulsion culture: A miniaturized library screening system based on micro-droplets in an emulsified medium
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.
Research highlights► Development of a novel culture method in a micro-compartment, “Emulsion Culture.” ► Functional signal peptides of beta-galactosidase in yeast were selected. ► Microchannel emulsification was applied to Emulsion Culture.
Journal: Journal of Bioscience and Bioengineering - Volume 112, Issue 3, September 2011, Pages 299–303