US4801529A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Methods for isolating mutant microoganisms using microcapsules coated with indicator material
Assignee: Brandeis University
•
Filing: 1985-06-18
•
Granted: 1989-01-31
•
Expires: 2005-06-18
Abstract
Abstract A method is disclosed for isolating a mutant microorganism. The method includes the following steps: (1) separately microencapsulating individual or small numbers of a microorganism population containing the mutant to obtain a first microdroplet; (2) thereafter surrounding the first microdroplet, with or without an outer semi-permeable membrane, with an outer gel-coating to obtain a gel-coated microdroplet, wherein the gel layer contains an indicator material which reacts to the presence of the mutant microorganism; (3) culturing the resultant microcapsules so that the microdroplets containing mutant microorganism are able to induce a detectable difference in the indicator material from microdroplets, containing non-mutant microorganisms; and (4) separating the microdroplets containing mutant microorganisms from those containing non-mutant microorganisms based upon the differences in the indicator material.
Inventors
- 👤 Daniel Perlman
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-04 05:45
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/5005; C; C12; C12Q; C12Q1/00; C12Q1/02; C12Q1/04
Claims Analysis
Total Claims: 32
Claim 1
Independent Claim
112 words
A method for isolating a mutant microorganism, where the method comprises: (a) separately microencapsulating individual or a small number of microorganisms from a microorganism population containing the mutant to obtain a first microdroplet; (b) surrounding the first microdroplet, thereby forming an inner microdroplet, with an outer gel-coating to obtain a gel-coated microdroplet, wherein the gel-coating on the microdroplet contains an indicator materials which reacts to the presence of the mutant microorganism; (c) incubating the gel-coated microdroplets to induce a detectable difference in the indicator material surrounding microdroplets containing mutant microorganisms; and (d) separating the microdroplets containing mutant microorganisms from those containing non-mutant microorganisms based upon the detectable difference in the indicator material.
Claim 2
Independent Claim
112 words
A method for isolating a mutant microorganism, where the method comprises: (a) separately microencapsulating individual or a small number of microorganisms from a microorganism population containing the mutant to obtain a first microdroplet; (b) surrounding the first microdroplet, thereby forming an inner microdroplet, with an outer gel-coating to obtain a gel-coated microdroplet, wherein the gel-coating on the microdroplet contains an indicator materials which reacts to the presence of the mutant microorganism; (c) incubating the gel-coated microdroplets to induce a detectable difference in the indicator material surrounding microdroplets containing mutant microorganisms; and (d) separating the microdroplets containing mutant microorganisms from those containing non-mutant microorganisms based upon the detectable difference in the indicator material.
Claim 3
Independent Claim
104 words
A method for isolating a mutant microorganism, where the method comprises: (a) separately microencapsulating an indicator material which reacts to the presence of the mutant microorganism to obtain a first microdroplet; (b) surrounding the microdroplet with an outer gel-coating to obtain a gel-coated microdroplet, wherein the gel-coating contains individual or a small number of microorganisms from a microorganism population containing the mutant; (c) incubating the gel-coated microdroplets so that the microdroplets containing mutant microorganisms induce a detectable difference in the indicator material; and (d) separating the microdroplets having mutant microorganisms from those having non-mutant microorganisms based upon the detectable difference in the indicator material.
Claim 4
Independent Claim
104 words
A method for isolating a mutant microorganism, where the method comprises: (a) separately microencapsulating an indicator material which reacts to the presence of the mutant microorganism to obtain a first microdroplet; (b) surrounding the microdroplet with an outer gel-coating to obtain a gel-coated microdroplet, wherein the gel-coating contains individual or a small number of microorganisms from a microorganism population containing the mutant; (c) incubating the gel-coated microdroplets so that the microdroplets containing mutant microorganisms induce a detectable difference in the indicator material; and (d) separating the microdroplets having mutant microorganisms from those having non-mutant microorganisms based upon the detectable difference in the indicator material.
Claim 5
Dependent Claim
18 words
The method of claim 1 wherein the indicator material reacts with a metabolite produced by the mutant microorganism.
Claim 6
Dependent Claim
29 words
The method of claim 1 wherein the indicator material reacts with a product formed by a reaction between the mutant microorganism and a substance included in its culture medium.
Claim 7
Dependent Claim
67 words
The method of claim 1, wherein the inner microdroplets containing the single microorganisms are formed by: (a) forming a dilute suspension of said microorganisms in a liquid diluent capable of forming a gel upon subsequent treatment, said dilute suspension having a dilution selected so that there is a high probability that each microdroplet produced from said suspension contains one microorganism; (b) converting said suspension into gel droplets.
Claim 8
Dependent Claim
62 words
The method of claim 5, wherein the outer gel coating is formed by: (a) forming a dilute suspension of indicator material in a liquid diluent capable of forming a gel upon subsequent treatment; (b) coating the first microdroplet with the suspension from (a); (c) ejecting the coated microdroplets into a solution which causes the coating to gel, resulting in a gel-coated microcapsule.
Claim 9
Dependent Claim
27 words
The method of claim 1, wherein a semi-permeable membrane is formed around the inner microdroplet by cross-linking surface layers of the inner microdroplet resulting in a microcapsule.
Claim 10
Dependent Claim
29 words
The method of claim 7, wherein a semi-permeable membrane is formed around the outer gel-coating by cross-linking surface layers of said outer gel-coating resulting in a double membrane microcapsule.
Claim 11
Dependent Claim
26 words
The method of claim 7, wherein the indicator material is a species of cells whose growth is affected by a metabolic product of the mutant microorganism.
Claim 12
Dependent Claim
37 words
The method of claim 1 wherein before coating the first microdroplet with an outer gel-coating, the microdroplet is cultured for a sufficient time to enable the individual or small number of microorganisms to multiply and form microcolonies.
Claim 13
Dependent Claim
38 words
The method of claim 1 where after coating the first microdroplet with an outer gel-coating, the gel-coated microdroplet is cultured for a sufficient time to enable the individual or small number of microorganisms to multiply and form microcolonies.
Claim 14
Dependent Claim
39 words
The method of claim 1, wherein said separation is based on the difference in mass of indicator material between said microdroplets containing mutant and non-mutant microorganisms and comprises equilibrium density centrifugation or sedimentation of a suspension containing said microdroplets.
Claim 15
Dependent Claim
39 words
The method of claim 9, wherein said separation is based on the difference in mass of indicator material between said microcapsules containing mutant and non-mutant microorganisms and comprises equilibrium density centrifugation or sedimentation of a suspension containing said microdroplets.
Claim 16
Dependent Claim
31 words
The method of claim 1, wherein the step of separating the microdroplets containing mutant from non-mutant microorganisms comprises fluorescent, colorimetric, or radioactive sorting of microdroplets in a suspension containing said microdroplets.
Claim 17
Dependent Claim
34 words
The method of claim 1, wherein the separation based on the difference in indicator material between said microdroplets containing mutant and non-mutant microorganisms comprises electrical or magnetic sorting of a suspension containing said microdroplets.
Claim 18
Dependent Claim
11 words
The method of claim 1, wherein said microorganisms comprise eucaryotic cells.
Claim 19
Dependent Claim
11 words
The method of claim 1, wherein said microorganisms comprise procaryotic cells.
Claim 20
Dependent Claim
10 words
The method of claim 1, wherein said microorganisms comprise viruses.
Claim 21
Dependent Claim
18 words
The method of claim 1, wherein said microorganisms comprise cell hybrids formed by fusing cells of different species.
Claim 22
Dependent Claim
29 words
The method of claim 1, wherein mutant microorganisms are incubated in a medium selected from the group consisting of chemically complex, non-sterile, agricultural, industrial, and other commercial process media.
Claim 23
Dependent Claim
40 words
The method of claim 9 wherein said growth is effected by amplifying the detectable difference between the indicator species in microcapsules containing the mutant microorganisms and the non-mutant microorganisms, and the separating step is based on physical characteristics amplified thereby.
Claim 24
Dependent Claim
16 words
The method of claim 1, wherein the microencapsulated microorganisms are grown in a laboratory culturing medium.
Claim 25
Dependent Claim
32 words
The method of claim 7, wherein the inner microencapsulated microorganisms are grown in a non-sterile, multi-species agricultural or industrial process medium wherein one or more species are found in the native environment.
Claim 26
Dependent Claim
19 words
The method of claim 1, wherein the mutant microorganism is a naturally occurring mutant of a wild type microorganism.
Claim 27
Dependent Claim
15 words
The method of claim 1, wherein the mutation in the mutant microorganism is artificially induced.
Claim 28
Dependent Claim
22 words
The method of claim 25, wherein the artificially-induced mutant is the result of genetic engineering by a biological, biochemical or biophysical process.
Claim 29
Dependent Claim
13 words
The method of claim 1 wherein the indicator material is a non-living material.
Claim 30
Dependent Claim
28 words
The method of claim 27 wherein the non-living indicator material is selected from the group consisting of labelled or unlabelled antibodies, antigens, enzymes, enzyme substrates and combinations thereof.
Claim 31
Dependent Claim
54 words
The method of claim 9, wherein said metabolic product of the mutant microorganism is an essential metabolite for said indicator species, and the microcapsules are incubated under conditions restrictive to growth of said indicator species, whereby the microcapsules containing the mutants have a greater number of indicator cells per microcapsule than microcapsules containing non-mutants.
Claim 32
Dependent Claim
56 words
The method of claim 9, wherein said metabolic product of the mutant microorganism is an inhibitor of the growth of said indicator species, and the microcapsules are incubated under conditions permissive for growth of said indicator species, whereby the microcapsules containing the mutants have a less number of indicator cells per microcapsules than microcapsules containing non-mutants.
References Cited (Backward Citations)
US3551295A
Cited
US3856628A
Cited
US4255411A
Cited
US4352883A
Cited
US4391909A
Cited
US4409331A
Cited
US4278761A
Cited
US4401762A
Cited
US4401755A
Cited
US4407957A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1992-01-31 | 1992-08-01 | 1993-01-31 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1996-01-31 | 1996-08-01 | 1997-01-31 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2000-01-31 | 2000-08-01 | 2001-01-31 | $7700.00 | Window Closed / Lapsed |