# US 4,399,219 A · Process for isolating microbiologically active material

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 4,399,219 A
- Document ID: US4399219A
- Status: Granted
- Kind: A
- Application number: 06/229,484
- Filed: 1981-01-29
- Granted: 1983-08-16
- Earliest claimed: 1981-01-29
- Assignees: Massachusetts Institute of Technology
- Inventors: James C. Weaver
- Web page: https://patentgrep.com/patent/US4399219A

## CPC

- C12N 11/04: entrapped within the carrier, e.g. gel or hollow fibres
- C12N 1/02: Separating microorganisms from their culture media
- C12N 9/00: Enzymes; Proenzymes; Compositions thereof (preparations containing enzymes for cleaning teeth A61K8/66, A61Q11/00; medicinal preparations containing enzymes or proenzymes A61K38/43; enzyme containing detergent compositions C11D; {enzymes with nucleic acid structure, e.g. ribozymes, C12N15/113}); Processes for preparing, activating, inhibiting, separating or purifying enzymes (preparation of malt C12C1/00)
- C12Q 1/00: Measuring or testing processes involving enzymes, nucleic acids or microorganisms (measuring or testing apparatus with condition measuring or sensing means, e.g. colony counters, C12M1/34); Compositions therefor; Processes of preparing such compositions
- Y10S 435/808: Optical sensing apparatus
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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
- Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

## Abstract

Molecules or cells of materials exhibiting a microbiological activity, suppression or interaction of interest are isolated from a large population of similar molecules, cells or the like. The activity of the microbiologically active molecules or living matter of interest is measured by suspending the population in a liquid medium capable of forming a gel. The resultant suspension is formed into droplets which are caused to gel. The gel droplets (GMDs) are treated to effect desirable alteration of the microbiologically active material and the amount of metabolites or reaction products of the desired alteration within each of the gel droplets. Alternatively, incubation is carried out such that each GMD initially containing one cell contains many, a microcolony, which can be tested for desirable properties while retaining sufficient viable cells for further growth and harvesting for further tesing and/or production. The gel droplets are then separated based upon the measured or sensed characteristic, metabolite or reaction product.

## Claims

### Claim 1

The process of selecting molecules or cells having a desired microbiological activity of a sample of a given material comprising the steps of:
(a) forming mutually independent samples of said material by
(i) forming a dilute suspension of said material in a liquid diluent capable of forming a gel upon subsequent treatment, said dilute suspension having a dilution selected so that there is provided a high probability that each microsample produced from said suspension contains one or less microbiologically active molecule or cell;
(ii) converting said suspension into gel droplets having a size between about 0.2 and 1000 microns,
(iii) measuring a product of microbiological activity of each of said gel droplets independently of the other of said droplets and
(b) separating gel droplets displaying the desired microbiological activity.

### Claim 2 (depends on 1)

The process of claim 1 wherein said droplets have a size between about 5 and 100 microns.

### Claim 3 (depends on 1)

The process of claim 1 wherein said material is a bacterium.

### Claim 4 (depends on 3)

The process of claim 3 wherein said bacterium is E. coli.

### Claim 5 (depends on 4)

The process of claim 4 wherein said bacterium includes a genetically modified plasmid.

### Claim 6 (depends on 1)

The process of claim 1 wherein said material is an enzyme.

### Claim 7 (depends on 1)

The process of claim 1 wherein said material is a yeast.

### Claim 8 (depends on 1)

The process of claim 1 wherein said material is a mold.

### Claim 9 (depends on 1)

The process of claim 1 wherein said material comprises an animal cell.

### Claim 10 (depends on 1)

The process of claim 1 wherein said material comprises a plant cell.

### Claim 11 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10)

The process of any one of claims 1, 2, 3, 4, 6, 7, 8, 9, 10 or 5 wherein the microbiologically active material having the desired characteristic is selectively tagged with a marker composition capable of being sensed and sensing said marker composition of effect isolation of gel droplets containing the microbiologically active material having the desired characteristic.

### Claim 12 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10)

The process of any one of claims 1, 2, 3, 4, 6, 7, 8, 9, 10 or 5 wherein the microbiologically active material having the desired characteristic produces a metabolite capable of being sensed and sensing said metabolite to effect isolation of gel micro-droplets containing the microbiologically active material having the desired characteristic.

### Claim 13 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10)

The process of any one of claims 1, 2, 3, 4, 6, 7, 8, 9, 10 or 5 wherein the microbiologically active material having the desired characteristic coacts with a reagent within said mciro-droplet to product a reaction product capable of being sensed and sensing said reaction product to effect isolation of gel micro-droplets containing the microbiologically active material having the desired characteristic.

### Claim 14

The process for selectively isolating gel microdroplets containing a cell or molecule having microbiological activity having a desired microbiological characteristic from a large population of microbiologically active cells or molecules lacking said desired characteristic which comprises forming a dilute suspension of said population in a liquid diluent capable of forming a gel upon subsequent treatment, said dilution being selected so that there is provided a high probability that each sub-sample produced from said suspension contains one or less microbiologically active molecule or cell, converting said suspension into gel droplets having a size between about 0.2 microns and about 1000 microns sensing the gel droplets containing the microbiologically active material having the desired characteristic and selectively separating said gel droplets containing microbiologically active material having the desired characteristic from said population.

### Claim 15 (depends on 14)

The process of claim 14 wherein said material is a bacterium.

### Claim 16 (depends on 15)

The process of claim 15 wherein said bacterium is E. coli.

### Claim 17 (depends on 16)

The process of claim 16 wherein said bacterium includes a genetically modified plasmid.

### Claim 18 (depends on 14)

The process of claim 14 wherein said material is an enzyme.

### Claim 19 (depends on 14)

The process of claim 14 wherein said material is a yeast.

### Claim 20 (depends on 14)

The process of claim 14 wherein said material is a mold.

### Claim 21 (depends on 14)

The process of claim 14 wherein the material comprises an animal cell.

### Claim 22 (depends on 14)

The process of claim 14 wherein the material comprises a plant cell.

### Claim 23 (depends on 14, 15, 16, 17, 18, 19, 20, 21, 22)

The process of any one of claims 14, 15, 16, 18, 19, 20, 21, 22 or 17 wherein the microbiologically active material having the desired characteristic is selectively tagged with a marker composition capable of being sensed and sensing said marker composition to effect isolation of gel droplets containing the microbiologically active material having the desired characteristic.

### Claim 24 (depends on 14, 15, 16, 17, 18, 19, 20, 21, 22)

The process of any one of claims 14, 15, 16, 18, 19, 20, 21, 22 or 17 wherein the microbiologically active material having the desired characteristic produces a metabolite capable of being sensed and sensing said metabolite to effect isolation of gel micro-droplets containing the microbiologically active material having the desired characteristic.

### Claim 25 (depends on 14, 15, 16, 17, 18, 19, 20, 21, 22)

The process of any one of claims 14, 15, 16, 18, 19, 20, 21, 22 or 17 wherein the microbiologically active material having the desired characteristic coacts with a reagent within said mciro-droplet to produce a reaction product capable of being sensed and sensing said reaction product to effect isolation of gel micro-droplets containing the microbiologically active material having the desired characteristic.