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FACS-sorting libraries

Droplets for FACS-sorting libraries

 

Microfluidic droplets can behave as compartments that link genotype to some otherwise unlinked phenotypes (e.g. activities of secreted proteins; fluorogenic assays on enzymes from lysed cells; an effect on another cell). Furthermore, microfluidic droplets can be sorted on conventional Fluorescence Activated Cell Sorting (FACS) machines, by turning them into double emulsion droplets (i.e. water droplets in an oil ‘shell’, in an aqueous continuous phase).

FACS-sorting encapsulated libraries via double emulsion (DE) droplet technology enables useful applications: in vitro evolution (e.g. of viruses); directed evolution of proteins from expression libraries, such as therapeutic proteins, DNA/RNA polymerases with novel or modified activities, or industrial enzymes.

  • In vitro evolution, e.g., of viruses, expression libraries, IVTT clones
    Protein engineering by directed evolution
  • Expression libraries, in vitro transcription, etc.
  • Millions of individual cells can be encapsulated with a reaction mix, and quantitatively sorted based on a read-out reaction
  • The biocompatible emulsion oil is compatible with reactions
  •  The encapsulation oil allows gas exchange, so cells can be cultured for days

Dolomite Bio droplet systems and chips allow DE formation using a reliable two-step process, recently published in a leading journal. First, water-in-oil droplets are formed, and the experimental work takes place. The emulsion is then reinjected and formation of the oil shell takes place (see Double emulsions application note). This stage can be done at the most convenient time – typically just before FACS analysis.
Double emulsions can be formed quickly and easily using the µEncapsulator system.

Watch the μEncapsulator Video

Zinchenko, et al. “One in a million: flow cytometric sorting of single cell-lysate assays in monodisperse picolitre double emulsion droplets for directed evolution.” Analytical Chemistry. 86.5 (2014): 2526-2533.

Larsen, et al. “A general strategy for expanding polymerase function by droplet microfluidics.” Nature communications 7 (2016).

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