US4760022A
Expired / Lapsed
US
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Stabilized plasmids
Assignee: Takeda Pharma AS
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Filing: 1983-09-15
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Granted: 1988-07-26
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Expires: 2003-09-15
Abstract
Abstract PCT No. PCT/DK83/00086 Sec. 371 Date May 15, 1984 Sec. 102(e) Date May 15, 1984 PCT Filed Sep. 15, 1983 PCT Pub. No. WO84/01172 PCT Pub. Date Mar. 29, 1984.Plasmids which are in themselves unstably inherited or which have become unstable due to the insertion of a DNA fragment comprising one or more genes not naturally related to the plasmid are stablized by means of a partitioning function exerted by a par region, especially a plasmid R1 par region, inserted into the plasmid on a DNA fragment which may be the length of the wild-type R1 EcoR1-. A fragment, but which is preferably shorter than this fragment, and which may comprise the R1 par region A, the R1 par region B or both these R1 par regions. The stabilization obtained for several different types of plasmid, especially by employing both R1 par regions, approaches the stability level of wild-type plasmids, i.e. they typically have a frequency of loss of less than 5x1031 6 per cell per generation. Such stabilized plasmids are useful in large-scale production of gene products as no particular bacterial strains or mutants are needed to secure plasmid maintenance, and as is not necessary to employ a specific composition of the nutrient medium in which the host cells are grown in order to prevent loss of the plasmid from the bacterial population.
Inventors
- 👤 Soen Molin
- 👤 Kenn A. Gerdes
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-24 02:22
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/67; C12N15/69; C12N15/68
Claims Analysis
Total Claims: 29
Claim 1
Independent Claim
64 words
A plasmid which replicates in gram negative bacteria and which has been stabilized by the insertion of a DNA fragment consisting essentially of par region A, par region B or both par region A and par region B of plasmid R1, said DNA fragment having a length under 19 kilobase pairs, the plasmid comprising a gene or genes not naturally related to the plasmid.
Claim 2
Dependent Claim
64 words
A plasmid which replicates in gram negative bacteria and which has been stabilized by the insertion of a DNA fragment consisting essentially of par region A, par region B or both par region A and par region B of plasmid R1, said DNA fragment having a length under 19 kilobase pairs, the plasmid comprising a gene or genes not naturally related to the plasmid.
Claim 3
Dependent Claim
32 words
The plasmid according to claim 1, wherein the inserted DNA fragment comprises both R1 par region A and R1 par region B, said fragment having a length not exceeding about 6 kb.
Claim 4
Dependent Claim
27 words
The plasmid according to claim 1, wherein the inserted DNA fragment comprises the R1 par region A, said fragment having a length not exceeding about 4 kb.
Claim 5
Dependent Claim
27 words
The plasmid according to claim 1, wherein the inserted DNA fragment comprises the R1 par region B, said fragment having a length not exceeding about 2 kb.
Claim 6
Dependent Claim
13 words
The plasmid according to claim 1, further comprising a gene mediating antibiotic resistance.
Claim 7
Dependent Claim
36 words
The plasmid according to claim 1, wherein the plasmid is selected from the group consisting of a p15 plasmid or a derivative thereof, and a high copy number broad host range plasmid or a derivative thereof.
Claim 8
Dependent Claim
24 words
The plasmid of claim 1, wherein said plasmid is selected from the group consisting of a pMB1 plasmid, a pBR322 plasmid, and derivatives thereof.
Claim 9
Dependent Claim
25 words
The plasmid of claim 1, wherein said plasmid has a low copy number during least one stage in the cultivation of bacteria harboring the plasmid.
Claim 10
Dependent Claim
16 words
The plasmid of claim 8, wherein said low copy number is about 0.5-5 copies per cell.
Claim 11
Dependent Claim
42 words
The plasmid of claim 8, wherein said plasmid is selected from the group consisting of (a) plasmids of the incompatibility group IncFII, (b) F plasmids, (c) low copy number broad host range plasmids, and (d) derivatives of plasmids, a, b, or c.
Claim 12
Dependent Claim
14 words
The plasmid of claim 8, wherein said plasmid is a conditional runaway replication plasmid.
Claim 13
Dependent Claim
67 words
The plasmid of claim 11, wherein said plasmid has a copy number not exceeding about 3-5 copies per cell when host microorganisms containing the plasmid are grown under conditions which insure a low plasmid copy number, and a copy number in the range of at least about 500-1000 copies per cell when the host microorganisms are grown under different conditions insuring a substantially increased plasmid copy number.
Claim 14
Dependent Claim
56 words
The plasmid of claim 12, wherein said plasmid has a copy number not exceeding about 3-5 copies per cell at a first temperature at which host microorganisms are grown, and a plasmid copy number in the range of at least about 500-1000 copies per cell at a second higher temperature at which host microorganisms are grown.
Claim 15
Dependent Claim
30 words
The plasmid of claim 12, wherein said plasmid copy number is in the range of about 0.5-1 copy per cell under the conditions which insure a low plasmid copy number.
Claim 16
Dependent Claim
16 words
The plasmid of claim 12, wherein said plasmid is a plasmid of the incompatibility group IncFII.
Claim 17
Dependent Claim
10 words
A bacterium which harbors a plasmid according to claim 1.
Claim 18
Dependent Claim
12 words
The bacterium of claim 16 wherein said bacterium is an Escherichia coli.
Claim 19
Dependent Claim
35 words
A method for producing a gene product of plasmid DNA, comprising cultivating a bacterium harboring the plasmid of claim 1 for production of a gene product, and harvesting said gene product from the bacterial culture.
Claim 20
Dependent Claim
18 words
The method of claim 18, wherein the bacterial cultivation proceeds for at least 100 generations of the bacteria.
Claim 21
Dependent Claim
24 words
The method of claim 19, wherein the frequency of loss factor of the plasmid from the host cell is less than 2×10 -4 /cell/generation.
Claim 22
Dependent Claim
25 words
The method according to claim 20, wherein the frequency of loss factor of the plasmid from the host cell is less than 10 -5 /cell/generation.
Claim 23
Dependent Claim
24 words
The method of claim 21, wherein the frequency of loss factor of the plasmid from the host cell is less than 5×10 -6 /cell/generation.
Claim 24
Independent Claim
78 words
A method of screening for the correct insertion of a par region in a plasmid, comprising transforming a bacterium already harboring an unrelated plasmid carrying the same par region as well as mediating a phenotype which is recognizable on appropriate media, with a plasmid believed to carry an inserted par region, culturing said transformed bacterium on said media, and evaluating said culture for the loss of said phenotype as indicative of the correct insertion of the par region.
Claim 25
Independent Claim
78 words
A method of screening for the correct insertion of a par region in a plasmid, comprising transforming a bacterium already harboring an unrelated plasmid carrying the same par region as well as mediating a phenotype which is recognizable on appropriate media, with a plasmid believed to carry an inserted par region, culturing said transformed bacterium on said media, and evaluating said culture for the loss of said phenotype as indicative of the correct insertion of the par region.
Claim 26
Dependent Claim
13 words
The method according to claim 23, wherein the recognizable phenotype is antibiotic resistance.
Claim 27
Dependent Claim
14 words
The method according to claim 23, wherein the recognizable phenotype is Lac + .
Claim 28
Dependent Claim
16 words
The method according to claim 23, wherein the par region on said unrelated plasmid is parA.
Claim 29
Dependent Claim
16 words
The method according to claim 24, wherein the par region on said unrelated plasmid is parB.
References Cited (Backward Citations)
US4487835A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1991-07-26 | 1992-01-25 | 1992-07-26 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1995-07-26 | 1996-01-25 | 1996-07-26 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1999-07-26 | 2000-01-25 | 2000-07-26 | $7700.00 | Window Closed / Lapsed |