In vitro compartmentalization by double emulsions: sorting and gene enrichment by fluorescence activated cell sorting.

Citation:

Bernath K, Hai M, Mastrobattista E, Griffiths AD, Magdassi S, Tawfik DS. In vitro compartmentalization by double emulsions: sorting and gene enrichment by fluorescence activated cell sorting. Anal. Biochem.Analytical Biochemistry. 2004;325 (1) :151 - 157.

Date Published:

2004///

Abstract:

Water-in-oil (w/o) emulsions can be used to compartmentalize and select large gene libraries for a predetd. function. The aq. droplets of the w/o emulsion function as cell-like compartments in each of which a single gene is transcribed and translated to give multiple copies of the protein (e.g., an enzyme) it encodes. While compartmentalization ensures that the gene, the protein it encodes, and the products of the activity of this protein remain linked, it does not directly afford a way of selecting for the desired activity. Here the authors show that re-emulsification of w/o emulsions gives water-in-oil-in-water (w/o/w) emulsions with an external (continuous) water phase through which droplets contg. fluorescent markers can be isolated by fluorescence-activated cell sorting (FACS). These w/o/w emulsions can be sorted by FACS, while the content of the aq. droplets of the primary w/o emulsion remains intact. Consequently, genes embedded in these water droplets together with a fluorescent marker can be isolated and enriched from an excess of genes embedded in water droplets without a fluorescent marker. The ability of FACS instruments to sort up to 40,000 events per s may endow this technol. a wide potential in the area of high-throughput screening and the directed evolution of enzymes. [on SciFinder(R)]