Methods for in vitro sorting of molecular and cellular libraries, such as a gene library, that are microencapsulated using water-in-oil-in-water emulsions.

Citation:

Tawfik D, Bernath K, Aharoni A, Peisajovich S, Griffiths AD, Mastrobattista E, Magdassi S.; 2005. Methods for in vitro sorting of molecular and cellular libraries, such as a gene library, that are microencapsulated using water-in-oil-in-water emulsions.

Abstract:

The present invention provides an in vitro system for compartmentalization of mol. or cellular libraries and provides methods for selection and isolation of desired mols. or cells from the libraries. The library includes a plurality of distinct mols. or cells encapsulated within a water-in-oil-in-water (w/o/w) emulsion. The emulation includes a continuous external aq. phase and a discontinuous dispersion of water-in-oil droplets. The internal aq. phase of a plurality of such droplets comprises a specific mol. or cell that is within the plurality of distinct mols. or cells of the library. According to a first aspect the present invention provides a gene library comprising a plurality of re-emulsified water-in-oil droplets, each droplet comprises an external water phase surrounding a central water-in-oil droplet, the internal water phase within each droplet comprises a genetic element, in vitro transcription-translation reaction system. To ensure that the genetic elements and gene products may not diffuse between primary water-in-oil droplets or between re-emulsified water-in-oil droplets, the-contents of each droplet must be isolated from the contents of the surrounding droplets, so that there is no or little exchange of gene products between the droplets over the timescale of the expt. The method of the present invention requires that there are only a limited no. of genetic elements per droplet. This ensures that the gene product of an individual genetic element will be isolated from other genetic elements. Finally, the formation and the compn. of the droplets must not interrupt with the function of the expression machinery of the genetic elements and the activity of the gene products. Prepn. and sorting of w/o/w emulsions by FACS (fluorescence-activated cell sorting) were demonstrated using lacZ reporter selection from a pool of lacZ gene mutants. Compartmentalization and detection of PON1 (serum paraoxonase) gene variants in single Escherichia coli cells were also demonstrated. [on SciFinder(R)]