US4374927A
Expired / Lapsed
US
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Extrachromosomal regulation of expression
Assignee: Leland Stanford Junior University
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Filing: 1981-02-24
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Granted: 1983-02-22
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Expires: 2001-02-24
Abstract
Abstract Microbiological methods, compositions and transformants are used for production of organic products for controlling cellular properties. Extrachromosomal elements are used which are subject to external modulation for production of a control element. The change in the amount of production of the control element allows for enhanced expression of a gene producing a poly(amino acid) product. The change in production of the control element allowing for enhanced gene expression of the product is accompanied by amplification of the product producing gene.
Inventors
- 👤 John J. Sninsky
- 👤 Stanley N. Cohen
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-01 22:15
CPC Classifications
C; C12; C12N; C12N15/00
Claims Analysis
Total Claims: 37
Claim 1
Independent Claim
177 words
A method for producing a poly(amino acid) product on demand, said method comprising: (a) growing to a high density transformant microorganism host cells having at least one extrachromosomal element capable of replication in said host cells which includes: (1) an activatable runaway-replication vector, (2) components of a first regulatory system comprising a first regulatory element subject to control by an external modulator, wherein said first regulatory element regulates a gene producing a control factor, (3) components of a second regulatory system comprising a second regulatory element, subject to control by said control factor, wherein said second regulatory element regulates a gene producing a poly(amino acid) product, (4) a gene expressing a poly(amino acid) product replicated in conjunction with said runaway-replication-vector, (5) a gene expressing a control factor; (b) subjecting said cells to an external modulator which modulates expression of said control factor producing gene in a direction enhancing expression of said product producing gene, while activating said runaway-replication vector, where enhanced expression and amplification of said product producing gene occurs with enhanced production of said poly(amino acid).
Claim 2
Independent Claim
177 words
A method for producing a poly(amino acid) product on demand, said method comprising: (a) growing to a high density transformant microorganism host cells having at least one extrachromosomal element capable of replication in said host cells which includes: (1) an activatable runaway-replication vector, (2) components of a first regulatory system comprising a first regulatory element subject to control by an external modulator, wherein said first regulatory element regulates a gene producing a control factor, (3) components of a second regulatory system comprising a second regulatory element, subject to control by said control factor, wherein said second regulatory element regulates a gene producing a poly(amino acid) product, (4) a gene expressing a poly(amino acid) product replicated in conjunction with said runaway-replication-vector, (5) a gene expressing a control factor; (b) subjecting said cells to an external modulator which modulates expression of said control factor producing gene in a direction enhancing expression of said product producing gene, while activating said runaway-replication vector, where enhanced expression and amplification of said product producing gene occurs with enhanced production of said poly(amino acid).
Claim 3
Dependent Claim
17 words
A method according to claim 1, wherein said runaway-replication vector is activated by a change in temperature.
Claim 4
Dependent Claim
15 words
A method according to claim 2, wherein said external modulator is a change in temperature.
Claim 5
Dependent Claim
17 words
A method according to claim 2, wherein said external modulator is a low molecular weight organic compound.
Claim 6
Independent Claim
22 words
A method according to any of claims 1, 2 or 3, wherein said control factor inhibits expression of said product producing gene.
Claim 7
Dependent Claim
21 words
A method according to claim 5, wherein said control factor is a repressor and said second regulatory element is an operator.
Claim 8
Independent Claim
22 words
A method according to any of claims 1, 2 or 3, wherein said control factor stimulates expression of said product producing gene.
Claim 9
Independent Claim
43 words
A method according to any of claims 1, 2 or 3 where said first and second regulatory elements are on separate extrachromosomal elements, and also included is a replicon which regulates replication of said first regulatory element and said control factor producing gene.
Claim 10
Independent Claim
22 words
A method according to any of claims 1, 2 or 3, wherein said poly(amino acid) inhibits the growth of said host cell.
Claim 11
Independent Claim
22 words
A method according to any of claims 1, 2 or 3, wherein said poly(amino acid) is rapidly degraded in said host cells.
Claim 12
Independent Claim
181 words
A method for producing a poly(amino acid) product on demand, said method comprising: (a) growing to a high density transformant microorganism host cells having two extrachromosomal elements capable of replication in said host cell, wherein a first extrachromosomal element includes: (1) components of a first regulatory system comprising a first regulatory element subject to control by an external modulator, wherein said first regulatory element regulates a gene expressing a control factor; and (2) a gene expressing a control factor; and a second extrachromosomal element includes: (1) an activatable runaway-replication vector; (2) components of a second regulatory system comprising a second regulatory element subject to control by said control factor, wherein said second regulatory element regulates a gene expressing said poly(amino acid) product; and (3) a gene expressing said poly(amino acid) product; (b) subjecting said cells to said external modulator which modulates expression of said control factor producing gene in a direction enhancing expression of said product producing gene, while activating said runaway-replication vector, where enhanced expression and amplification of said product producing gene occurs with enhanced production of said poly(amino acid).
Claim 13
Independent Claim
181 words
A method for producing a poly(amino acid) product on demand, said method comprising: (a) growing to a high density transformant microorganism host cells having two extrachromosomal elements capable of replication in said host cell, wherein a first extrachromosomal element includes: (1) components of a first regulatory system comprising a first regulatory element subject to control by an external modulator, wherein said first regulatory element regulates a gene expressing a control factor; and (2) a gene expressing a control factor; and a second extrachromosomal element includes: (1) an activatable runaway-replication vector; (2) components of a second regulatory system comprising a second regulatory element subject to control by said control factor, wherein said second regulatory element regulates a gene expressing said poly(amino acid) product; and (3) a gene expressing said poly(amino acid) product; (b) subjecting said cells to said external modulator which modulates expression of said control factor producing gene in a direction enhancing expression of said product producing gene, while activating said runaway-replication vector, where enhanced expression and amplification of said product producing gene occurs with enhanced production of said poly(amino acid).
Claim 14
Dependent Claim
27 words
A method according to claim 11, wherein said runaway-replication vector is activated by a change in temperature and said external modulator is a parallel change in temperature.
Claim 15
Independent Claim
21 words
A method according to any of claims 11 or 12, wherein said control factor inhibits expression of said product producing gene.
Claim 16
Dependent Claim
21 words
A method according to claim 13, wherein said control factor is a repressor and said second regulatory element is an operator.
Claim 17
Independent Claim
18 words
A method according to any of claims 11 or 12, wherein said poly(amino acid) product is an enzyme.
Claim 18
Dependent Claim
107 words
A microorganism cell having at least one extrachromosomal element capable of replication in said cell which includes: (1) an activatable runaway-replication vector; (2) components of a first regulatory system comprising a first regulatory element subject to control by an external modulator, wherein said first regulatory element regulates a gene producing a control factor; (3) components of a second regulatory system comprising a second regulatory element subject to control by said control factor, wherein said second regulatory element regulates a gene producing a poly(amino acid) product; (4) a gene expressing said poly(amino acid) product replicated in conjunction with said runaway-replication vector, (5) a gene expressing a control factor.
Claim 19
Dependent Claim
107 words
A microorganism cell having at least one extrachromosomal element capable of replication in said cell which includes: (1) an activatable runaway-replication vector; (2) components of a first regulatory system comprising a first regulatory element subject to control by an external modulator, wherein said first regulatory element regulates a gene producing a control factor; (3) components of a second regulatory system comprising a second regulatory element subject to control by said control factor, wherein said second regulatory element regulates a gene producing a poly(amino acid) product; (4) a gene expressing said poly(amino acid) product replicated in conjunction with said runaway-replication vector, (5) a gene expressing a control factor.
Claim 20
Dependent Claim
27 words
A cell according to claim 16, wherein said runaway-replication vector is activated by a change in temperature and said external modulator is a parallel change in temperature.
Claim 21
Dependent Claim
29 words
A cell according to any of claims 16 or 17, wherein (2) is on a first extrachromosomal element and (1), (3) and (4) are on a second extrachromosomal element.
Claim 22
Dependent Claim
12 words
A cell according to claim 18, wherein said control factor inhibits expression.
Claim 23
Dependent Claim
13 words
A cell according to claim 18, wherein said poly(amino acid) is an enzyme.
Claim 24
Independent Claim
200 words
A method for producing a poly(amino acid) product on demand, said method comprising: (a) growing to a high density transformant microorganism host cells having at least one extrachromosomal element capable of replication in said host cells which include: (1) an activatable runaway-replication vector; (2) components of a first regulatory system comprising a first regulatory element regulating a gene producing a control factor; (3) a gene expressing a control factor; (4) components of a second regulatory system comprising a second regulatory element, subject to control by said control factor, wherein said second regulatory element regulates a gene producing a poly(amino acid) product wherein said control factor is a component of said second regularoty system; (5) a gene expressing a poly(amino acid) product replicated in conjunction with said runaway-replication vector; wherein at least one of said first and second regulatory systems is subject to control by an external modulator; (b) subjecting said cells to an external modulator which modulates at least one of said first and second regulatory systems in a direction enhancing expression of said product producing gene, while activating said runaway-replication vector, wherein enhanced expression and amplification of said product producing gene occurs with enhanced production of said poly(amino acid).
Claim 25
Independent Claim
200 words
A method for producing a poly(amino acid) product on demand, said method comprising: (a) growing to a high density transformant microorganism host cells having at least one extrachromosomal element capable of replication in said host cells which include: (1) an activatable runaway-replication vector; (2) components of a first regulatory system comprising a first regulatory element regulating a gene producing a control factor; (3) a gene expressing a control factor; (4) components of a second regulatory system comprising a second regulatory element, subject to control by said control factor, wherein said second regulatory element regulates a gene producing a poly(amino acid) product wherein said control factor is a component of said second regularoty system; (5) a gene expressing a poly(amino acid) product replicated in conjunction with said runaway-replication vector; wherein at least one of said first and second regulatory systems is subject to control by an external modulator; (b) subjecting said cells to an external modulator which modulates at least one of said first and second regulatory systems in a direction enhancing expression of said product producing gene, while activating said runaway-replication vector, wherein enhanced expression and amplification of said product producing gene occurs with enhanced production of said poly(amino acid).
Claim 26
Dependent Claim
15 words
A method according to claim 21, wherein said external modulator controls said second regulatory system.
Claim 27
Dependent Claim
12 words
A method according to claim 22, wherein said external modulator is temperature.
Claim 28
Dependent Claim
21 words
A method according to claim 22, wherein said control factor is a repressor and said second regulator element is an operator.
Claim 29
Dependent Claim
18 words
A method according to claim 24, wherein at least one of said repressor and operator is temperature sensitive.
Claim 30
Dependent Claim
12 words
A method according to claim 25, wherein said repressor is expressed constitutively.
Claim 31
Dependent Claim
118 words
A microorganism cell having at least one extrachromosomal element capable of replication in said cell which include: (1) an activatable runaway-replication vector; (2) components of a first regulatory system comprising a first regulatory element which regulates a gene producing a control factor; (3) components of a second regulatory system comprising a second regulatory element subject to control by said control factor, wherein said second regulatory element regulates a gene producing a poly(amino acid) product, and said second regulatory system includes said control factor; (4) a gene expressing said poly(amino acid) product; (5) a gene expressing said control factor; wherein at least one component of said first and second regulatory systems is subject to control by an external modulator.
Claim 32
Dependent Claim
118 words
A microorganism cell having at least one extrachromosomal element capable of replication in said cell which include: (1) an activatable runaway-replication vector; (2) components of a first regulatory system comprising a first regulatory element which regulates a gene producing a control factor; (3) components of a second regulatory system comprising a second regulatory element subject to control by said control factor, wherein said second regulatory element regulates a gene producing a poly(amino acid) product, and said second regulatory system includes said control factor; (4) a gene expressing said poly(amino acid) product; (5) a gene expressing said control factor; wherein at least one component of said first and second regulatory systems is subject to control by an external modulator.
Claim 33
Dependent Claim
21 words
A cell according to claim 27, wherein said control factor is a repressor and said second regulatory element is an operator.
Claim 34
Dependent Claim
16 words
A cell according to any of claims 27 or 28, wherein said external modulator is temperature.
Claim 35
Dependent Claim
26 words
A cell according to claim 27, wherein (2) and (5) are on one extrachromosomal element and (1), (3) and (4) are on a second extrachromosomal element.
Claim 36
Dependent Claim
20 words
A cell according to claim 30, wherein said external modulator controls at least one component of said second regulatory system.
Claim 37
Dependent Claim
12 words
A cell according to claim 31, wherein said external modulator is temperature.
References Cited (Backward Citations)
DD467A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1986-02-21 | 1986-08-23 | 1987-02-22 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1990-02-21 | 1990-08-23 | 1991-02-22 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1994-02-21 | 1994-08-23 | 1995-02-22 | $7700.00 | Window Closed / Lapsed |