Patent US2525447A successfully refreshed from Google Patents.
Patent EP0109150A2 successfully refreshed from Google Patents.
EP0109150A2
Expired / Lapsed
EP
⏱️ 0.0 years remaining
Plasmids with conditional uncontrolled replication behaviour
Assignee: Takeda Pharma AS
•
Filing: 1983-09-16
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Granted: —
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Expires: 2003-09-16
Abstract
Abstract Plasmids are disclosed which carry a regulatable promoter which has been inserted in such a way that transcription from the promoter regulates replication so that 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 λP R , the activity of which is controlled by a temperature-sensitive λcl repressor. Three types of plasmid are disclosed: one in which λP R replaces part of one native replication regulatory gene of the plasmid leading to a plasmid copy number of about 20-40 copies per cell at about 30 'C and an uncontrolled plasmid copy number in the range of at least about 500-1000 copies per cell at about 36-42 'C (a «type A plasmid (Fig. 7)); another plasmid in which λP R has been inserted upstream of the native replication control genes of the plasmid leading to a plasmid copy number of about 3-5 copies per cell at about 30 °C and an uncontrolled plasmid copy number in the range of at least about 500-1000 copies per cell at about 42 °C (a «type 8 plasmid. (Fig. 8)); and a third plasmid constructed from a type A plasmid in which the deleted native replication gene has been inserted outside the replication region of the plasmid leading to a plasmid copy number of about 0.5-1 copy per cell at about 30 °C and an uncontrolled plasmid copy number in the range of at least about 500-1000 copies per cell at about 42 * C (a «type C plasmid. (Fig. 9)). The plasmids may be used as cloning vectors for the insertion of a fragment of foreign, e.g. eucaryotic, DNA coding for a desired polypeptide. To obtain gene products of the plasmids, host microorganisms to which plasmids of any of the above-mentioned types have been transformed are cultivated at about 30 °C to secure a constant, low plasmid copy number during the seeding and multiplication stages of the microorganism, after which the temperature is shifted to about 36-42 °C to obtain an uncontrolled plasmid copy number and an amplified amount of gene product. This latter temperature may be maintained until the cells cease to grow, or it may, after a period of time sufficient to obtain a substantial amplification of plasmid DNA, be lowered to the temperature at which the plasmid copy number is constant to secure the continued growth of the host cells while still obtaining amplified amounts of gene product. The gene product is then harvested from the culture.
Inventors
- 👤 Soren Molin
- 👤 Janice Ann Light
- 👤 Jens Erik Love Larsen
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-25 11:36
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/67; C12N15/68; C12N15/69
Claims Analysis
Total Claims: 74
Claim 1
Independent Claim
21 words
A plasmid in which a regulatable promoter has been inserted in such a way that transcription from this promoter regulates replication.
Claim 2
Dependent Claim
21 words
A plasmid in which a regulatable promoter has been inserted in such a way that transcription from this promoter regulates replication.
Claim 3
Dependent Claim
35 words
A plasmid in which a regulatable promoter has been inserted in such a way that transcription from this promoter regulates replication so that increased transcription leads to a substantially increased or uncontrolled plasmid copy number.
Claim 4
Dependent Claim
35 words
A plasmid in which a regulatable promoter has been inserted in such a way that transcription from this promoter regulates replication so that increased transcription leads to a substantially increased or uncontrolled plasmid copy number.
Claim 5
Dependent Claim
30 words
A plasmid according to claim 1 or 2 in which the promoter is one which is inherently regulatable due to a particular DNA structure within the region of the promoter.
Claim 6
Dependent Claim
20 words
A plasmid according to claim 1 or 2 in which the promoter is controllable by means of a regulating factor.
Claim 7
Dependent Claim
17 words
A plasmid according to claim 4 in which the regulating factor is one that exerts positive control.
Claim 8
Dependent Claim
17 words
A plasmid according to claim 4 in which the regulating factor is one that exerts negative control.
Claim 9
Dependent Claim
31 words
A plasmid according to claim 2 in which increased transcription from the regulatable promoter leading to a substantially increased or uncontrolled plasmid copy number is caused by derepression of said promoter.
Claim 10
Dependent Claim
19 words
A plasmid according to any of the preceding claims in which the promoter is one which is chemically regulatable.
Claim 11
Dependent Claim
19 words
A plasmid according to claim 8 in which the promoter is a lac promoter, trp promoter or deo promoter.
Claim 12
Dependent Claim
23 words
A plasmid according to any claims 1-7 in which the activity of the promoter is temperature-dependent or controlled by a temperature-sensitive regulating factor.
Claim 13
Dependent Claim
33 words
A plasmid according to claim 10 in which the promoter is a X promoter, and in which the gene for a temperature-sensitive λ cl repressor controlling transcription from said promoter is also present.
Claim 14
Dependent Claim
19 words
A plasmid according to claim 11 in which the X promoter is either λP R or λP L .
Claim 15
Dependent Claim
15 words
A plasmid according to claim 12 in which the \ promoter is λP R .
Claim 16
Dependent Claim
40 words
A plasmid according to any of the preceding claims 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 17
Dependent Claim
44 words
A plasmid according to claim 14 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 18
Dependent Claim
17 words
A plasmid according to claim 15 showing an uncontrolled plasmid copy number at temperatures above about 39°C.
Claim 19
Dependent Claim
27 words
A plasmid according to any of the preceding claims from which part of one native replication regulatory gene has been deleted and replaced by the regulatable promoter.
Claim 20
Dependent Claim
77 words
A plasmid according to claim 17 which, when host micro-organisms containing the plasmid are grown under conditions securing a constant, low plasmid copy number, has a copy number in the range of 20-40, preferably 20-30, and particularly 20-25 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 21
Dependent Claim
64 words
A plasmid according to claim 18 which has a plasmid copy number in the range of about 20-40, preferably 20-30 and particularly 20-25 copies per cell at one temperature, such as a temperature of about 30°C, and an uncontrolled plasmid copy number in the range of at least about 500-1000 copies per cell at a higher temperature, such as a temperature of about 42°C.
Claim 22
Dependent Claim
27 words
A plasmid according to any of claims 17-19 which is an Rl-type plasmid from which the copB gene has been deleted and replaced by the regulatable promoter.
Claim 23
Dependent Claim
70 words
A plasmid 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 about 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
70 words
A plasmid 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 about 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 25
Dependent Claim
59 words
A plasmid according to claim 21 which has a plasmid copy number in the range of about 3-5 copies per cell at one temperature, such as a temperature of about 30°C, and an uncontrolled plasmid copy number in the range of at least about 500-1000 copies per cell at a higher temperature, such as a temperature of about 42°C.
Claim 26
Dependent Claim
26 words
A plasmid according to any of claims 1-16 in which a regulatable promoter has been inserted upstream of the native replication control gene(s) of the plasmid.
Claim 27
Dependent Claim
73 words
A plasmid according to claim 23 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 about 3-5 copies per cell, and which, when the host micro-organisms 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 28
Dependent Claim
59 words
A plasmid according to claim 24 which has a plasmid copy number in the range of about 3-5 copies per cell at one temperature, such as a temperature of about 30°C, and an uncontrolled plasmid copy number in the range of at least about 500-1000 copies per cell at a higher temperature, such as a temperature of about 42°C.
Claim 29
Dependent Claim
28 words
A plasmid according to any of claims 23-25 which is an RI-type plasmid in which the regulatable promoter has been inserted upstream of the copB and copA genes.
Claim 30
Dependent Claim
69 words
A plasmid 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 about 0.5-1 copy 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 31
Dependent Claim
69 words
A plasmid 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 about 0.5-1 copy 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 32
Dependent Claim
59 words
A plasmid according to claim 27 which has a plasmid copy number .in the range of about 0.5-1 copy per cell at one temperature, such as a temperature of about 30°C, and an uncontrolled plasmid copy number in the range of at least about 500-1000 copies per cell at a higher temperature, such as a temperature of about 42°C.
Claim 33
Dependent Claim
49 words
A plasmid according to any of claims 1-16 which carries a regulatable promoter 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 where it is not naturally located.
Claim 34
Dependent Claim
74 words
A plasmid according to claim 29 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 about 0.5-1 copy 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 35
Dependent Claim
59 words
A plasmid according to claim 30 which has a plasmid copy number in the range, of about 0.5-1 copy per cell at one temperature, such as a temperature of about 30°C, and an uncontrolled plasmid copy number in the range of at least about 500-1000 copies per cell at a higher temperature, such as a temperature of about 42°C.
Claim 36
Dependent Claim
32 words
A plasmid according to any of claims 29-31 which is an Rl-type plasmid in which the copB gene has been inserted at the EcoRI site outside the replication region of the plasmid.
Claim 37
Dependent Claim
50 words
A plasmid according to any of the preceding claims in which, in addition to the first regulatable promoter regulating transcription from the replication region, a second promoter has been inserted in a manner permitting the insertion of a structural gene the expression of which is controlled from said second promoter.
Claim 38
Dependent Claim
19 words
A plasmid according to claim 33 in which the second promoter regulating the expression of said gene is controllable.
Claim 39
Dependent Claim
26 words
A plasmid according to claim 33 or 34 in which said second inserted promoter controlling the expression of the structural gene is the λP L promoter.
Claim 40
Dependent Claim
47 words
A plasmid according to any of claims 33-35 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 41
Dependent Claim
22 words
A plasmid according to claim 36 having an uncontrolled plasmid copy number and a very high gene expression at temperatures above 39°C.
Claim 42
Dependent Claim
24 words
A plasmid according to any of the preceding claims which carries a marker useful for the identification and/or selection of cells containing the plasmid.
Claim 43
Dependent Claim
20 words
A plasmid according to claim 38 in which the marker is a gene mediating antibiotic resistance to the host microorganism.
Claim 44
Dependent Claim
30 words
A plasmid according to any of the preceding claims which carries a gene allowing gene fusion upon insertion of another gene from which fusion it is possible to detect expression.
Claim 45
Dependent Claim
16 words
A plasmid according to any of the preceding claims, characterized in that it is a miniplasmid.
Claim 46
Dependent Claim
22 words
A plasmid according to any of the preceding claims which additionally carries a gene or genes not naturally related to the plasmid.
Claim 47
Dependent Claim
13 words
A microorganism in which a plasmid according to claims 1-42 has -been inserted.
Claim 48
Dependent Claim
10 words
A microorganism according to claim 43 which is a bacterium.
Claim 49
Dependent Claim
13 words
A microorganism according to claim 44 which is a gram-negative and mesophilic bacterium.
Claim 50
Dependent Claim
11 words
A microorganism according to claim 45 which is an Escherichia coli.
Claim 51
Independent Claim
57 words
A method of constructing a plasmid according to claims 1-42 comprising inserting a regulatable promoter into a plasmid in such a way that transcription from said promoter regulates replication and identifying the plasmid so treated by screening for those plasmids which show a substantially increased or uncontrolled plasmid copy number when transcription from said promoter is increased.
Claim 52
Dependent Claim
35 words
A method according to claim 47 comprising deleting part of one native replication regulatory gene and inserting a DNA fragment including the regulatable promoter at the site of the partially deleted native replication regulatory gene.
Claim 53
Dependent Claim
33 words
A method according to claim 48 comprising deleting the copB gene from the plasmid by cleaving with the restriction endonuclease BglII and inserting a Bglll fragment including the regulated promoter at this site.
Claim 54
Dependent Claim
21 words
A method according to claim 47 comprising inserting the regulatable promoter into the plasmid upstream of the native replication control gene(s).
Claim 55
Dependent Claim
23 words
A method according to claim 50 comprising inserting the regulatable promoter at the Bglll site upstream of the copB and copA genes. -
Claim 56
Dependent Claim
46 words
A method according to claim 47 comprising, by using the plasmid with the partially deleted native replication regulatory gene as a starting material, reinserting the native replication regulatory gene at its original site to construct a plasmid in which all native replication control genes are present.
Claim 57
Dependent Claim
44 words
A method according to claim 52 comprising, by using the plasmid with the partially deleted copB gene as a starting material, reinserting the copB gene at the Bglll site to construct a plasmid in which both the copB and the copA genes are present.
Claim 58
Dependent Claim
41 words
A method according to claim 47 comprising, by using the plasmid with the partially deleted native replication regulatory gene as a starting material, inserting one native replication regulatory gene in a region of the plasmid where it is not naturally located.
Claim 59
Dependent Claim
36 words
A method according to claim 54 comprising, by using the plasmid with the partially deleted copB gene as a starting material, inserting the copB gene at the EcoRl site outside the replication region of the plasmid.
Claim 60
Dependent Claim
48 words
A method of constructing a plasmid according to claim 33 comprising, in addition to the insertion of the regulatable promoter, the insertion of said second promoter at an appropriate restriction site permitting the insertion of a structural gene the expression of which is regulatable from said second promoter.
Claim 61
Independent Claim
59 words
A method of producing a gene product of plasmid DNA which comprises cultivating microorganisms carrying a plasmid according to claims 1-42 comprising 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 62
Dependent Claim
65 words
A method according to claim 57 comprising cultivating the host microorganism, to which a plasmid according to claims 1-42 has been transformed, 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 then cultivating the host micro-organism under conditions leading to a substantially increased or uncontrolled plasmid copy number.
Claim 63
Dependent Claim
57 words
A method according to claim 57 or 58 in which, after a period of cultivation under conditions where any transcription from the promoter does not significantly influence the copy number of the plasmid, a substance inducing promoter activity is added to the culture medium in an amount leading to a substantially increased or uncontrolled plasmid copy number.
Claim 64
Dependent Claim
72 words
A method according to claim 59 in which, after maintaining the 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, the substance inducing promoter activity is removed from the culture medium leading to a slow decrease in plasmid copy number to the pre-induction level.
Claim 65
Dependent Claim
50 words
A method according to claim 57 or 58 in which, after a period of cultivation in the presence of a substance substantially inhibiting promoter activity, the concentration of the substance is reduced to an extent leading to activation of the promoter and a substantially increased or uncontrolled plasmid copy number.
Claim 66
Dependent Claim
68 words
A method according to claim 61 in which, after 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, the substance inhibiting promoter activity is added in an amount leading to a slow decrease in plasmid copy number to pre-induction level. "
Claim 67
Dependent Claim
41 words
A method according to claim 57 or 58 wherein the conditions under which the host microorganism is cultivated comprise at least a period of cultivation at or near a temperature at which the plasmid copy number is substantially increased or uncontrolled.
Claim 68
Dependent Claim
46 words
A method according to claim 63 wherein the cultivation at the temperature at which the plasmid copy number is substantially increased or uncontrolled is continued until the growth of the host microorganism is inhibited by the formation of plasmid DNA or gene products from the plasmids.
Claim 69
Dependent Claim
69 words
A method according to claim 63 wherein production scale cultivation is performed continuously at a temperature approaching the temperature at which the plasmid copy number is substantially increased or uncontrolled so as to permit the survival and continual growth of the host microorganism concomitantly with formation of increased amounts of a gene product of the plasmid, and the gene product is then continuously or intermittently harvested from the culture.
Claim 70
Dependent Claim
107 words
A method according to claim 63 wherein, after the propagation of the microbial culture to reach production size, the temperature is shifted to a temperature at or near the temperature at which the plasmid copy number is substantially increased or uncontrolled, and this temperature is maintained 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, after which the temperature is once more 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 71
Independent Claim
38 words
A method according to any of claims 63-66 wherein the temperature at which the plasmid copy number is substantially increased or uncontrolled is higher than the temperature at which the plasmid has a constant, low copy number. ""
Claim 72
Dependent Claim
47 words
A method according to claim 67 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 73
Independent Claim
31 words
A method for preparing a plasmid according to any of claims 1-42 comprising inserting a DNA fragment carrying a marker useful for the identification and/or selection of cells containing the plasmid.
Claim 74
Dependent Claim
20 words
A method according to claim 69 in which the marker is a gene mediating antibiotic resistance to the host microorganism.
References Cited (Backward Citations)
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