# US 2017/0282133 A1 · In vitro evolution in microfluidic systems

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- Number: US 2017/0282133 A1
- Document ID: US20170282133A1
- Status: Published application
- Kind: A1
- Application number: 15/588,210
- Filed: 2017-05-05
- Published: 2017-10-05
- Earliest claimed: 2004-10-08
- Assignees: Medical Research Council; President and Fellows of Harvard College
- Applicants: Medical Research Council; President and Fellows of Harvard College
- Inventors: Andrew David Griffiths; David A. Weitz; Darren R. Link; Keunho Ahn; Jerome Bibette
- Web page: https://patentgrep.com/patent/US20170282133A1

## CPC

- B01F 23/41: Emulsifying
- 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
- B01F 25/4331: {Mixers with bended, curved, coiled, wounded mixing tubes or comprising elements for bending the flow}
- B01F 25/4338: {Mixers with a succession of converging-diverging cross-sections, i.e. undulating cross-section}
- 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
- 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}
- B01F 33/3031: using electro-hydrodynamic [EHD] or electro-kinetic [EKI] phenomena to mix or move the fluids
- 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)}
- B01L 3/502746: {characterised by the means for controlling flow resistance, e.g. flow controllers, baffles or throttle valves}
- B01L 3/502753: {characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation}
- B01L 3/502776: {specially adapted for focusing or laminating flows}
- B01L 3/502784: {specially adapted for droplet or plug flow, e.g. digital microfluidics}
- 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}
- C12N 15/1075: {by coupling phenotype to genotype, not provided for in other groups of this subclass}
- C12P 21/00: Preparation of peptides or proteins (single cell protein C12N1/00)
- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- G01N 15/1459: {the analysis being performed on a sample stream}
- G01N 15/1484: {microstructural devices}
- B01J 2219/00466: in a slurry
- B01J 2219/00468: by manipulation of individual beads
- B01J 2219/005: Beads
- B01J 2219/0052: in the shape of elongated tubes
- B01J 2219/00545: Colours
- B01J 2219/00576: fluorophore
- B01J 2219/00657: One-dimensional arrays
- B01J 2219/00722: Nucleotides
- B01L 2200/0636: Focussing flows, e.g. to laminate flows
- B01L 2200/0673: Handling of plugs of fluid surrounded by immiscible fluid
- B01L 2300/0654: Lenses; Optical fibres
- B01L 2300/0816: Cards, e.g. flat sample carriers usually with flow in two horizontal directions
- B01L 2300/0864: comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
- B01L 2300/0867: Multiple inlets and one sample wells, e.g. mixing, dilution
- B01L 2400/0415: electrical forces, e.g. electrokinetic
- B01L 2400/0487: fluid pressure, pneumatics
- G01N 15/149: specially adapted for sorting particles, e.g. by their size or optical properties
- B01F: MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING (mixing paints B44D3/06)
- B01J: CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## 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

### Claim at position 1

1-107. (canceled)

### Claim 108

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.

### Claim 109 (depends on 108)

The method of claim 108, wherein the genetic elements are nucleic acids, proteins, or cells.

### Claim 110 (depends on 109)

The method of claim 109, wherein the nucleic acids are primers for a polymerase chain reaction (PCR).

### Claim 111 (depends on 108)

The method of claim 108, wherein the genetic elements are labeled.

### Claim 112 (depends on 108)

The method of claim 108, wherein the microcapsules are monodisperse with respect to each other.

### Claim 113 (depends on 108)

The method of claim 108, 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.

### Claim 114 (depends on 108)

The method of claim 108, wherein the concentration of the beads is adjusted such that a single bead appears in each microcapsule.

### Claim 115 (depends on 108)

The method of claim 108, wherein the genetic element is identified with a tag.

### Claim 116 (depends on 109)

The method of claim 109, wherein the genetic element is an RNA molecule.

### Claim 117 (depends on 108)

The method of claim 108, wherein the droplet generator comprises a channel that narrows relative to the direction of flow.

### Claim 118 (depends on 108)

The method of claim 108, wherein the enzymatic reaction is an amplification reaction.

### Claim 119 (depends on 118)

The method of claim 118, wherein the amplification reaction is a strand displacement amplification.

### Claim 120 (depends on 107)

The method of claim 107, wherein the microcapsules are produced using a flow-focusing geometry.

### Claim 121 (depends on 108)

The method of claim 108, wherein the enzymatic reaction is a reverse transcription reaction.