In vitro evolution in microfluidic systems

• US 10,639,598 B2
• Filed: 03/05/2018
• Issued: 05/05/2020
• Est. Priority Date: 10/08/2004
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation), UK Research and Innovation
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Griffiths, Andrew David, Weitz, David A., Link, Darren Roy, Ahn, Keunho, Bibette, Jerome
• Primary Examiner(s): Bhat, Narayan K
• Application Number:
  US15/912,033
• Publication Number:
  US 20180272294A1
• Time in Patent Office: 792 Days
• CPC Class Codes: B01F 23/41 Emulsifying
• CPC Class Codes: B01F 25/433 Mixing tubes wherein the shape of the tube influences the mixing, e.g. mixing tubes with varying cross-section or provided with inwardly extending profiles
• CPC Class Codes: B01F 25/4331 Mixers with bended, curved, coiled, wounded mixing tubes or comprising elements for bending the flow
• CPC Class Codes: B01F 25/4338 Mixers with a succession of converging-diverging cross-sections, i.e. undulating cross-section
• CPC Class Codes: B01F 33/3011 using a sheathing stream of a fluid surrounding a central stream of a different fluid, e.g. for reducing the cross-section of the central stream or to produce droplets from the central stream
• CPC Class Codes: B01F 33/3021 the components to be mixed being combined in a single independent droplet, e.g. these droplets being divided by a non-miscible fluid or consisting of independent droplets
• CPC Class Codes: B01F 33/3031 using electro-hydrodynamic [EHD] or electro-kinetic [EKI] phenomena to mix or move the fluids
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00466 in a slurry
• CPC Class Codes: B01J 2219/00468 by manipulation of individual beads
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/0052 in the shape of elongated tubes
• CPC Class Codes: B01J 2219/00545 Colours
• CPC Class Codes: B01J 2219/00576 fluorophore
• CPC Class Codes: B01J 2219/00657 One-dimensional arrays
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01L 2200/0636 Focussing flows, e.g. to laminate flows
• CPC Class Codes: B01L 2200/0673 Handling of plugs of fluid surrounded by immiscible fluid
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0864 : comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2300/0867 : Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2400/0415 : electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 2400/0487 : fluid pressure, pneumatics
• CPC Class Codes: B01L 3/502746 : characterised by the means for controlling flow resistance, e.g. flow controllers, baffles B01L3/502738 takes precedence
• CPC Class Codes: B01L 3/502753 : characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation separation in general B01D; microapparatus for analysis using electrophoresis G01N27/44791; sample preparation G01N1/28
• CPC Class Codes: B01L 3/502776 : specially adapted for focusing or laminating flows
• CPC Class Codes: B01L 3/502784 : specially adapted for droplet or plug flow, e.g. digital microfluidics automatic analysis using a stream of discrete samples in a tube system G01N35/08
• CPC Class Codes: C12N 15/1058 : Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms
• CPC Class Codes: C12N 15/1075 : by coupling phenotype to genotype, not provided for in other groups of this subclass
• CPC Class Codes: C12P 21/00 : Preparation of peptides or proteins single cell protein C12N1/00
• CPC Class Codes: C12Q 1/6874 : involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: G01N 15/1459 : the analysis being performed on a sample stream
• CPC Class Codes: G01N 15/1484 : microstructural devices
• CPC Class Codes: G01N 15/149 : specially adapted for sorting particles, e.g. by their size or optical properties

Abstract

The invention describes a method for isolating one or more genetic elements encoding a gene product having a desired activity, comprising the steps of: (a) compartmentalising genetic elements into microcapsules; and (b) sorting the genetic elements which express the gene product having the desired activity; wherein at least one step is under microfluidic control. The invention enables the in vitro evolution of nucleic acids and proteins by repeated mutagenesis and iterative applications of the method of the invention.

Claims

1. A method for analyzing compounds, the method comprising:

providing a droplet generator comprising a nozzle through which aqueous fluid is flowed under microfluidic control into a reservoir of oil to form a plurality of aqueous microcapsules in the oil, wherein each aqueous microcapsule comprises a genetic element and a cell;

incubating the plurality of aqueous microcapsules to cause the genetic element to interact with a molecule associated with the cell;

conducting an enzymatic reaction involving the molecule associated with the cell within one or more of the aqueous microcapsules, wherein the genetic element or a gene product encoded by the genetic element has an effect on the enzymatic reaction, to produce reaction products corresponding to individual gene products or genetic elements; and

analyzing the individual genetic elements identified based on detection of a selectable change caused by the reaction products in one or more of the aqueous microcapsules.

2. The method of claim 1, wherein the plurality of aqueous microcapsules are unable to fuse or coalesce due to the presence of a surfactant.

3. The method of claim 1, wherein the selectable change comprises an optical signal.

4. The method of claim 1, wherein the genetic element comprises one or more primer sequences for amplification.

5. The method of claim 1, wherein the genetic element is attached to a bead.

6. The method of claim 1, wherein the molecule is a reporter protein.

7. The method of claim 1, wherein the molecule comprises a gene.

8. The method of claim 7, further comprising expressing the gene to form a gene product.

9. The method of claim 8, wherein the gene product is a protein.

10. The method of claim 9, wherein the protein is an antibody.

11. The method of claim 9, wherein the protein is located within the cell.

12. The method of claim 9, wherein the protein is located outside of the cell.

13. The method of claim 1, wherein the molecule is DNA or RNA.

14. The method of claim 9, wherein the gene product is cDNA.