US4806471A
Expired / Lapsed
US
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Plasmids with conditional uncontrolled replication behavior
Assignee: A KINGDOM OF DENMARK CORP AS
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Filing: 1984-05-16
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Granted: 1989-02-21
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Expires: 2004-05-16
Abstract
Abstract Plasmids useful as cloning vectors carrying an inserted regulatable promoter, transcription from the promoter regulating plasmid replication. Increased transcription leads to a substantially increased or uncontrolled plasmid copy number when host microorganisms containing the plasmid are cultivated under conditions securing such increased transcription. The regulatable promoter is preferably λ P R , the activity of which is controlled by a temperature-sensitive λ cl repressor. To obtain gene products of the plasmids, host microorganisms to which such plasmids have been transformed are desirably cultivated at about 30° C. to secure a constant, low plasmid copy number during the seeding and multiplication stages of the microorganisms, after which the temperature is preferably shifted to about 36°-42° C. to obtain an uncontrolled plasmid copy number and an amplified amount of gene product.
Inventors
- 👤 Soren Molin
- 👤 Janice A. Light
- 👤 Jens E. L. Larsen
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-24 18:16
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/67; C12N15/68; C12N15/69
Claims Analysis
Total Claims: 67
Claim 1
Independent Claim
47 words
A plasmid requiring transcription from a promoter for replication, and which carries an inserted regulatable promoter at a position and in an orientation so that transcription from the promoter regulates plasmid replication, increased transcription from the promoter leading to a substantial increased or uncontrolled plasmid copy number.
Claim 2
Dependent Claim
47 words
A plasmid requiring transcription from a promoter for replication, and which carries an inserted regulatable promoter at a position and in an orientation so that transcription from the promoter regulates plasmid replication, increased transcription from the promoter leading to a substantial increased or uncontrolled plasmid copy number.
Claim 3
Dependent Claim
13 words
The plasmid of claim 1, said plasmid capable of replicating in Escherichia coli.
Claim 4
Dependent Claim
18 words
A plasmid according to claim 1, in which the promoter is controllable by means of a regulating factor.
Claim 5
Dependent Claim
21 words
A plasmid according to claim 3, in which the regulating factor positively induces the promoter to increase transcription and plasmid replcation.
Claim 6
Dependent Claim
20 words
A plasmid according to claim 3, in which the regulating factor derepresses the promoter to increase transcription and plasmid replication.
Claim 7
Dependent Claim
16 words
A plasmid according to claim 1, in which the promoter is one which is chemically regulatable.
Claim 8
Dependent Claim
19 words
A plasmid according to claim 6, in which the promoter is a lac promoter, trp promoter or deo promoter.
Claim 9
Dependent Claim
15 words
A plasmid according to claim 1, in which the activity of the promoter is temperature-dependent.
Claim 10
Dependent Claim
20 words
A plasmid according to claim 1, in which the activity of the promoter is controlled by a temperature-sensitive regulating factor.
Claim 11
Dependent Claim
32 words
A plasmid according to claim 9, in which the promoter is a λ promoter, and in which the gene for a temperature-sensitive λcl repressor controlling transcription from said promoter is also present.
Claim 12
Dependent Claim
18 words
A plasmid according to claim 10, in which the promoter is either λP R or λP L .
Claim 13
Dependent Claim
14 words
A plasmid according to claim 11, in which the promoter is λP R .
Claim 14
Dependent Claim
37 words
A plasmid according to claim 1, in which transcription from said regulatable promoter causes the plasmid to show a constant, low copy number at low temperatures and a substantially increased or uncontrolled copy number at higher temperatures.
Claim 15
Dependent Claim
46 words
A plasmid according to claim 13, in which transcription from said regulatable promoter causes the plasmid to show a constant, low copy number at a temperature of about 30° C. and a substantially increased copy number at a temperature in the range of about 36°-42° C.
Claim 16
Dependent Claim
18 words
A plasmid according to claim 14 showing an uncontrolled plasmid copy number at temperatures above about 39° C.
Claim 17
Dependent Claim
24 words
A plasmid according to claim 1, from which part of one native replication regulatory gene has been deleted and replaced by the regulatable promoter.
Claim 18
Dependent Claim
69 words
A plasmid according to claim 16 which, when host microorganisms containing the plasmid are grown under conditions securing a constant, low plasid copy number, has a copy number in the range of 20-40, and which, when the host microorganisms are grown under different conditions leading to a substantially increased or uncontrolled plasmid copy number, has a copy number in the range of at least about 500-1000 copies per cell.
Claim 19
Dependent Claim
42 words
A plasmid according to claim 17, wherein the plasmid has a copy number in the range of 20-40 at one temperature and an increased or uncontrolled copy number in the range of at least 500-1000 copies per cell at a higher temperature.
Claim 20
Dependent Claim
50 words
A plasmic according to claim 18, wherein the plasmid has a copy number in the range of 20-40 at a temperature of about 30° C., and an increased or uncontrolled copy number in the range of at least about 500-1000 copies per cell at a temperature of about 42° C.
Claim 21
Dependent Claim
24 words
A plasmid accordingto claim 15 which is an R1-type plasmid from which the copB gene has been deleted and replaced by the regulatable promoter.
Claim 22
Dependent Claim
24 words
A plasmid according to claim 1, in which a regulatable promoter has been inserted upstream of the native replication control gene(s) of the plasmid.
Claim 23
Dependent Claim
72 words
A plasmid according to claim 21 which, when host microorganisms containing the plasmid are grown under conditions securing a constant, low plasmid copy number, has a copy number in the range of 3-5 copies per cell, and which when the host microorganisms are grown under different conditions leading to a substantially increased or uncontrolled plasmid copy number, has a copy number in the range of at least about 500-1000 copies per cell.
Claim 24
Dependent Claim
43 words
A plasmid according to claim 22, wherein the plasmid has a copy number in the range of 3-5 at one temperature and an increased or uncontrolled copy number in the range of at least about 500-1000 copies per cell at a higher temperature.
Claim 25
Dependent Claim
50 words
A plasmid according to claim 23, wherein the plasmid has a copy number in the range of 3-5 at a temperature of about 30° C., and an increased or uncontrolled copy number in the range of at least about 500-1000 copies per cell at a temperature of about 42° C.
Claim 26
Dependent Claim
26 words
A plasmid according to claim 23, which is an R1-type plasmid in which the regulatable promoter has been inserted upstream of the copB and copA genes.
Claim 27
Dependent Claim
52 words
A plasmic according to claim 1, wherein a regulatable promoter has been inserted in the replication region from which part of one native replication regulatory gene has been deleted, and in which the native replication regulatory gene has been inserted in a region of said plasmid where it is not naturally located.
Claim 28
Dependent Claim
70 words
A plasmid according to claim 26, which, when host micoorganisms are grown under conditions securing a constant, low plasmid copy number, has a copy number in a range of about 0.5-1 copy per cell, and which when the host micoorganisms are grown under different conditions leading to a substantially increased or uncontrolled plasmid copy number, has a copy number in the range of at least about 500-1000 copies per cell.
Claim 29
Dependent Claim
42 words
A plasmid according to cliam 27, wherein the plasmid has a copy number int he range of 0.5-1 at one temperature, and an increased or uncontrolled copy numberin the range of at least about 500-1000 copies per cell at a higher temperature.
Claim 30
Dependent Claim
42 words
A plasmid according to cliam 27, wherein the plasmid has a copy number int he range of 0.5-1 at one temperature, and an increased or uncontrolled copy numberin the range of at least about 500-1000 copies per cell at a higher temperature.
Claim 31
Dependent Claim
51 words
A plasmid according to to claim 28, wherein the plasmid has a copy number in the range of 0.5-1 at a temperature of about 30° C., and an increased or uncontrolled copy number in the range of at least about 500-1000 copies per cell at a temperature of about 42° C.
Claim 32
Dependent Claim
29 words
A plasmid according to claim 26, comprising an R1-type plasmid in which the copB gene has been inserted at the EcoRI site outside the replication region of the plasmid.
Claim 33
Dependent Claim
36 words
A plasmid according to claim 1, which in addition to the first regulatable promoter regulating transcription of the replication region, carries a second inserted promoter controlling the expression of a structural gene inserted in the plasmid.
Claim 34
Dependent Claim
19 words
A plasmid according to claim 31, in which the second promoter regulating the expression of said gene is controllable.
Claim 35
Dependent Claim
24 words
A plasmid according to claim 31, in which said second inserted promoter controlling the expression of the structural gene is the λP L promoter.
Claim 36
Dependent Claim
40 words
A plasmid according to claim 31 having a constant, low plasmid copy number and very little or no gene expression at one temperature and having a substantially increased or uncontrolled plasmid copy number and gene expression at a higher temperature.
Claim 37
Dependent Claim
47 words
A plasmid according to claim 34, having a constant, low plasmid copy number and very little or no gene expression at about 30° C., and having a substantially increased or uncontrolled plasmid copy number and gene expression at a temperature in the range of about 36°-42° C.
Claim 38
Dependent Claim
23 words
A plasmid according to claim 35 having an uncontrolled plasmid copy number and a very high gene expression at temperatures above 39° C.
Claim 39
Dependent Claim
19 words
A plasmid according to claim 1 which carries a marker useful for the selection of cells containing the plasmid.
Claim 40
Dependent Claim
20 words
A plasmid according to claim 37, in which the marker is a gene mediating antibiotic resistance to the host microorganism.
Claim 41
Dependent Claim
19 words
A plasmid according to claim 1 which additionally carries a gene or genes not naturally related to the plasmid.
Claim 42
Dependent Claim
13 words
A microorganism in which a plasmid according to claim 1 has been inserted.
Claim 43
Dependent Claim
10 words
A micoorganism according to claim 40 which is a bacterium.
Claim 44
Dependent Claim
13 words
A microorganism according to claim 41 which is a gram-negative and mesophilic bacterium.
Claim 45
Dependent Claim
11 words
A micoorganism according to claim 42 which is an Escherichia coli.
Claim 46
Dependent Claim
60 words
A method of constructing the plasmid of claim 1, comprising: (a) inserting a regulatable promoter into a plasmid at a position where transcription from said promoter regulates plasmid replication, and (b) identifying the plasmid so treated by screening for those plasmids whcih show a substantially increased or uncontrolled plasmid copy number under conditions wherein transcription from said promoter is increased.
Claim 47
Dependent Claim
37 words
A method according to claim 44, comprising: (a) deleting part of one native replication regulatory gene, and (b) inserting a DNA fragment including the regulatable promoter at the site of the partially deleted native replication regulatory gene.
Claim 48
Dependent Claim
35 words
A method according to claim 45, comprising: (a) deleting the copB gene from the plasmid by cleaving with the restriction endonuclease Bgl11, and (b) inserting a Bgl11 fragment including the regulated promoter at this site.
Claim 49
Dependent Claim
20 words
A method according to claim 44 comprising inserting the regulatable promoter into the plasmid upstream the native replication control gene(s).
Claim 50
Dependent Claim
21 words
A method according to claim 47 comprising inserting the regulatable promoter at the BglII site upstream of copB and copA genes.
Claim 51
Dependent Claim
34 words
A method according to claim 45, further comprisig reinserting the native replication regulatory gene at its original site in the plasmid to construct a plasmid in whic all native replication control genes are present.
Claim 52
Dependent Claim
34 words
A method according to claim 46, further comprising reinserting the copB gene at the Bgl11 site in the plasmid to construct a plasmid in which both the copB and the copA genes are present.
Claim 53
Dependent Claim
26 words
A method according to claim 45, further comprising inserting one native replication regulatory gene in a region of the plasmid where it is not naturally located.
Claim 54
Dependent Claim
23 words
A method according to claim 46, further comprisng inserting the copB gene at the EcoRI site outside the replication region of the plasmid.
Claim 55
Dependent Claim
61 words
A method of constructing the plasmid of claim 31, comprising (a) inserting a regulatable promoter into a plasmid at a position where transcription from said promoter regulates plasmic replication, and (b) inserting a second promoter at an appropriate restriction site in the plasmid, thereby permitting the insertion of a strucutural gene the expression of which is regulatable from said second promoter.
Claim 56
Dependent Claim
60 words
A method of producing a gene product of said plasmid DNA comprising: cultivating host micoorganisms carrying a plasmid according to claim 71 for at least a period of cultivation under conditions securing increased transcription from said regulatable promoter, resulting in a substantially increased or uncontrolled plasmid copy number, and harvesting, from the microbial culture, a gene product of the plasmid.
Claim 57
Dependent Claim
54 words
A method according to claim 54, comprising cultivating said host micoorganisms under conditions resulting in a constant, low plasmid copy number during the seeding and multiplication stages, until the desired number of cells has been obtained, and thereafter cultivating said host microorganisms under conditions leading to a substantially increased or uncontrolled plasmid copy number.
Claim 58
Dependent Claim
53 words
A method according to claim 54, comprising cultivating said host microorganisms under conditions where transcription from the promoter does not significantly influence the copy number of the plasmid, and thereafter adding a substance which induces promoter activity to the culture medium in an amount leading to a substantially increased or uncontrolled copy number.
Claim 59
Dependent Claim
70 words
A method according to claim 56, comprising maintaining conditions leading to a substantially increased or uncontrolled plasmid copy number for a period of time sufficient to obtain a substantial amplification of the plasmid, but sufficiently brief to avoid any substantial impairment of the microbial culture, and thereafter removing the substance inducing promoter activity from the culture medium resulting in a slow decrease in plasmid copy number to the pre-induction level.
Claim 60
Dependent Claim
44 words
A method according to claim 54, comprising cultivating said host microorganisms in the presence of a substance substantially inhibiting promoter activity, and thereafter reducing the concentration of said substance, resulting in activation of the promoter and a substantially increased or uncontrolled plasmid copy number.
Claim 61
Dependent Claim
66 words
A method according to claim 58, comprising maintaining the conditions leading to a substantially increased or uncontrolled copy number for a period sufficient to obtain a substantial amplification of the plasmid, but sufficiently brief to avoid any substantial impairment of the microbial culture, and thereafter adding the substance inhibiting promoter activity in an amount leading to a slow decrease in plasmid copy number to pre-induction level.
Claim 62
Dependent Claim
33 words
A method according to claim 54, wherein the cultivation conditions for securing increased transcription from the promoter comprise a temperature at or near which the plasmid copy number is substantially increased or uncontrolled.
Claim 63
Dependent Claim
32 words
A method according to claim 60, wherein said temperature is maintained until the growth of the host microorganism is inhibited by the formation of plasmid DNA or gene products from the plasmid.
Claim 64
Dependent Claim
39 words
A method according to claim 61, wherein said temperature is thereafter shifted to a temperature securing a low, constant plasmid copy number causing a slow decrease in the copy number of the plasmid until it reaches the initial level.
Claim 65
Dependent Claim
49 words
A method according to claim 62, wherein the temperature at which the plasmid has a constant, low copy number is a temperature of about 30° C., and the temperature at which the plasmid has a substantially increased or uncontrolled copy number is in the range of about 36°-42° C.
Claim 66
Dependent Claim
30 words
A method of preparing a plasmid according to claim 37, comprising inserting in said plasmid a DNA fagment carrying a marker useful for the selection of cells containing the plasmid.
Claim 67
Dependent Claim
20 words
A method according to claim 64, in which the marker is a gene mediating antibiotic resistance to the host micoorganism.
References Cited (Backward Citations)
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1992-02-21 | 1992-08-22 | 1993-02-21 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1996-02-21 | 1996-08-22 | 1997-02-21 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2000-02-21 | 2000-08-22 | 2001-02-21 | $7700.00 | Window Closed / Lapsed |