In vitro evolution in microfluidic systems

• US 9,919,277 B2
• Filed: 05/05/2017
• Issued: 03/20/2018
• 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: Medical Research Council (Government of United Kingdom), 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
• Application Number:
  US15/588,210
• Publication Number:
  US 20170282133A1
• Time in Patent Office: 319 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 conducting an enzymatic reaction, comprising the steps of:

providing a droplet generator to produce, under microfluidic control, a plurality of aqueous microcapsules surrounded by an immiscible continuous phase that comprises a fluorinated oil that comprises a fluorinated polymer surfactant, each of the plurality of microcapsules comprising an enzyme, a genetic element linked covalently or non-covalently to a bead, and reagents for the enzymatic reaction;

pooling the microcapsules into one or more common compartments such that a portion of the plurality of microcapsules contact each other but do not fuse with each other due to the presence of the surfactant; and

conducting the enzymatic reaction on the genetic element of at least one of the plurality of microcapsules within the one or more common compartments.

2. The method of claim 1, wherein the genetic element comprises nucleic acids.

3. The method of claim 2, wherein the nucleic acids further comprise primers for a polymerase chain reaction (PCR).

4. The method of claim 2, wherein the genetic element comprises RNA.

5. The method of claim 1, wherein the genetic element is labeled.

6. The method of claim 1, wherein the microcapsules are monodisperse with respect to each other.

7. The method of claim 1, wherein the droplet generator further comprises an aqueous fluid channel and two immiscible continuous phase channels and producing the plurality of microcapsules surrounded by the immiscible continuous phase under the microfluidic control comprises partitioning an aqueous fluid that is flowing through the aqueous fluid channel with two counter propagating streams of the immiscible continuous phase that are flowing through the immiscible continuous phase channels.

8. The method of claim 1, wherein the concentration of the beads is adjusted such that a single bead appears in each microcapsule.

9. The method of claim 1, wherein the genetic element is identified with a tag.

10. The method of claim 1, wherein the droplet generator comprises a channel that narrows relative to the direction of flow.

11. The method of claim 1, wherein the enzymatic reaction is an amplification reaction.

12. The method of claim 11, wherein the amplification reaction is a strand displacement amplification.

13. The method of claim 1, wherein the microcapsules are produced by infusing an aqueous fluid through a narrow constriction into a counter-propagating oil stream.

14. The method of claim 1, wherein the enzymatic reaction is a reverse transcription reaction.