Compositions and methods for in vitro sorting of molecular and cellular libraries

• US 20070077572A1
• Filed: 05/24/2006
• Published: 04/05/2007
• Est. Priority Date: 11/24/2003
• Status: Abandoned Application

• Current Assignee: Medical Research Council (Government of United Kingdom), Yissum Research Development Company of the Hebrew University of Jerusalem Ltd. (Hebrew University of Jerusalem), Yeda Research and Development Co. Ltd. (Weizmann Institute of Science)
• Original Assignee: Medical Research Council (Government of United Kingdom), Yissum Research Development Company of the Hebrew University of Jerusalem Ltd. (Hebrew University of Jerusalem), Yeda Research and Development Co. Ltd. (Weizmann Institute of Science)
• Inventors: Magdassi, Shlomo, Tawfik, Dan, Bernath, Kalia, Peisajovich, Sergio, Griffiths, Andrew, Aharoni, Amir, Mastrobattista, Enrico
• Application Number:
  US11/439,533
• Publication Number:
  US 20070077572A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 15/1075 by coupling phenotype to genotype, not provided for in other groups of this subclass
• CPC Class Codes: C40B 40/08 Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries

Abstract

The present invention provides an in vitro system for compartmentalization of molecular or cellular libraries and provides methods for selection and isolation of desired molecules or cells from the libraries. The library includes a plurality of distinct molecules or cells encapsulated within a water-in-oil-in-water emulsion. The emulsion includes a continuous external aqueous phase and a discontinuous dispersion of water-in-oil droplets. The internal aqueous phase of a plurality of such droplets comprises a specific molecule or cell that is within the plurality of distinct molecules or cells of the library.

Claims

1. A library comprising a plurality of distinct molecules encapsulated within a water-in-oil-in-water emulsion, the emulsion comprising a continuous external aqueous phase and a discontinuous dispersion of water-in-oil droplets, wherein the internal aqueous phase of a plurality of the droplets each comprises a specific molecule of said plurality of distinct molecules of the library.

2. The library of claim 1, wherein the specific molecule is selected from the group consisting of:

a genetic element, a protein, a carbohydrate and a small organic molecule.

3. The library of claim 2, wherein each said droplet further comprises at least one additional molecule capable of interacting with the specific molecule to generate a detectable signal.

4. The library of claim 3, wherein said droplet further comprises at least one molecule capable of modifying one or more optical properties of the droplets, the at least one molecule is selected from the group consisting of a fluorescent marker and a fluorogenic substrate.

5. The library of claim 3, wherein the specific molecule is an enzyme and the at least one additional molecule is a substrate of the enzyme.

6. The library of claim 1, wherein the specific molecule is an expressible genetic element and each droplet further comprises a reaction system for expressing said genetic element.

7. The library of claim 6, wherein the genetic element and its gene product are attached to each other by plasmid-display, ribosome-display, CIS display or mRNA-peptide fusion.

8. The library of claim 1, wherein said specific molecule is contained within an entity selected from the group consisting of:

a cell, a bacteriophage and a virus.

9. The library of claim 6, wherein the specific molecule is contained within a cell and the gene product of said specific molecule is obtained in a location selected from the group consisting of:

intracellular, extracellular, cellular surface, cellular cytoplasm and cellular periplasm.

10. The library of claim 8, said specific molecule is contained within a cell, wherein the plurality of cells each comprise a distinct expressible genetic element the genetic product thereof induces growth arrest, apoptosis or lysis.

11. A method for isolating or identifying one or more molecules having a desired function, comprising the steps of:

a) providing the library of claim 1, said library comprises molecules within a water-in-oil-in-water emulsion, the emulsion comprising an external aqueous phase and a discontinuous dispersion of a plurality of water-in-oil droplets, wherein the molecules are compartmentalized in the plurality droplets; and

b) screening the plurality of droplets for a molecule having a desired function.

12. The method of claim 11, wherein the screening step comprises identifying a change in the optical properties of a droplet of the plurality of droplets.

13. The method of claim 11, wherein the molecule is a genetic element encoding at least one gene product having a desired activity.

14. The method of claim 11, wherein each droplet of the plurality of droplets comprise at least one genetic element and in vitro transcription-translation reaction system.

15. The method of claim 14, wherein following step (a) the method comprises the step of expressing the genetic element to produce its gene product within the droplets.

16. The method of claim 15, wherein the gene product remains linked to its genetic element.

17. The method of claim 15, wherein the activity of the gene product results in the alteration of the expression of a second genetic element within the droplet and the activity of the gene product of the second genetic element enables the isolation of the first genetic element.

18. The method of claim 11, wherein the droplet comprises at least one molecule selected from the group consisting of a fluorescent marker and a fluorogenic substrate.

19. The method of claim 11, wherein screening of the droplets comprises a technique selected from the group consisting of flow cytometry, fluorescence microscopy, optical tweezers and micro-pipettes.

20. The method of claim 11, wherein said molecules are within cells such that the cells being compartmentalized in said plurality droplets.

21. The method of claim 11, wherein step (a) is a multistep process comprising the steps of:

(i) compartmentalizing molecules or cells into primary water-in-oil droplets; and

(ii) re-emulsifying the primary water-in-oil droplets of (i) with an external aqueous phase to obtain re-emulsified water-in-oil-in-water droplets.

22. The method of claim 11, further comprising isolating a sub-population of droplets comprising molecules encoding desired gene products.

23. The method of claim 22, wherein said molecules are pooled and subjected to mutagenesis.

24. The method of claim 23, further comprising re-compartmentalizing said molecules for further iterative rounds of screening.

25. The method of claim 22, wherein the desired gene products induce, directly or indirectly, a change in the optical properties of the droplets containing same, the change permitting the droplet to be sorted.

26. The method of claim 11, wherein step (a) comprises:

(a) compartmentalizing molecules into primary water-in-oil droplets, wherein the molecule are genetic elements;

(b) expressing the genetic elements to produce their respective gene products within the primary water-in-oil droplets; and

(c) re-emulsifying the primary water-in-oil droplets of (b) with an external aqueous phase to obtain a plurality of re-emulsified water-in-oil-in-water droplets.