US4626504A
Expired / Lapsed
US
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DNA transfer vector for gram-negative bacteria
Assignee: Mycogen Plant Science Inc
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Filing: 1983-07-01
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Granted: 1986-12-02
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Expires: 2003-07-01
Abstract
Abstract Modified E. coli vectors are disclosed that are mobilizable to a wide range of gram-negative bacteria, but are not self-transmissible. The modified E. coli plasmids replicate only in bacterial strains of the E. coli group. Mobilizer strains of E. coli are also provided, as well as methods for employing the modified E. coli vectors for transposon mutagenesis, site-specific gene transfer, and the construction of DNA libraries.
Inventors
- 👤 Alfred Puhler
- 👤 Reinhard Simon
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-24 16:05
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/74; C12N15/743
Claims Analysis
Total Claims: 23
Claim 1
Independent Claim
81 words
A suicide plasmid that is mobilizable, but not self-transmissible, comprising: (a) a first DNA segment containing a replicon functional in E. coli; and (b) a second DNA segment containing a Mob-site and oriT of a plasmid transferrable by conjugation in the presence of a tra function encoded by a gene that acts in trans and capable of transferring the plasmid to an organism in which said replicon does not function; said suicide plasmid not containing a gene providing said tra function.
Claim 2
Dependent Claim
81 words
A suicide plasmid that is mobilizable, but not self-transmissible, comprising: (a) a first DNA segment containing a replicon functional in E. coli; and (b) a second DNA segment containing a Mob-site and oriT of a plasmid transferrable by conjugation in the presence of a tra function encoded by a gene that acts in trans and capable of transferring the plasmid to an organism in which said replicon does not function; said suicide plasmid not containing a gene providing said tra function.
Claim 3
Dependent Claim
12 words
A DNA suicide plasmid according to claim 1 further comprising a transposon.
Claim 4
Dependent Claim
12 words
A DNA suicide plasmid according to claim 1 further comprising transposon Tn5.
Claim 5
Dependent Claim
14 words
A DNA suicide plasmid according to claim 1 comprising the Mob-site of plasmid RP4.
Claim 6
Dependent Claim
18 words
A suicide plasmid according to claim 1 wherein said replicon functional in E. coli is that of pACYC184.
Claim 7
Dependent Claim
18 words
A suicide plasmid according to claim 1 wherein said replicon functional in E. coli is that of pACYC177.
Claim 8
Dependent Claim
20 words
A suicide plasmid according to claim 1 wherein said replicon functional in the E. coli group is that of pBR325.
Claim 9
Dependent Claim
20 words
A suicide plasmid according to claim 1 selected from the group consisting of pSUP101, pSUP301, pSUP401, pSUP201, pSUP202, and pSUP203.
Claim 10
Dependent Claim
12 words
A suicide plasmid according to claim 1 further comprising a cos site.
Claim 11
Dependent Claim
12 words
A suicide plasmid according to claim 1 wherein said plasmid is pSUP205.
Claim 12
Dependent Claim
12 words
A bacterial cell containing and replicating the suicide plasmid of claim 1.
Claim 13
Dependent Claim
16 words
A bacterial cell according to claim 11 wherein said DNA suicide plasmid further comprises a transposon.
Claim 14
Dependent Claim
16 words
A bacterial cell according to claim 11 wherein said DNA suicide plasmid further comprises transposon Tn5.
Claim 15
Dependent Claim
18 words
A bacterial cell according to claim 11 wherein said DNA suicide plasmid comprises the Mob-site of plasmid RP4.
Claim 16
Dependent Claim
22 words
A bacterial cell according to claim 11 wherein said replicon functional in E. coli of said suicide plasmid is that of pACYC184.
Claim 17
Dependent Claim
22 words
A bacterial cell according to claim 11 wherein said replicon functional in E. coli of said suicide plasmid is that of pACYC177.
Claim 18
Dependent Claim
22 words
A bacterial cell according to claim 11 wherein said replicon functional in E. coli of said suicide plasmid is tht of pBR325.
Claim 19
Dependent Claim
26 words
A bacterial cell according to claim 11 wherein said DNA suicide plasmid is selected from the group consisting of pSUP101, pSUP301, pSUP401, pSUP201, pSUP202, and pSUP203.
Claim 20
Dependent Claim
16 words
A bacterial cell according to claim 11 wherein said suicide plasmid further comprises a cos site.
Claim 21
Dependent Claim
14 words
A bacterial cell according to claim 19 wherein said DNA suicide plasmid is pSUP205.
Claim 22
Dependent Claim
108 words
A method of site-specific mutagenesis in a gram-negative bacterial cell comprising the steps of: (a) transferring into said bacterial cell a composite DNA plasmid comprising the suicide plasmid of claim 1 further comprising a third DNA segment homologous with a corresponding fourth DNA segment of said bacterial cell, said third DNA segment containing a mutation and a selectable genetic marker; and (b) incubating said bacterial cell after said transfer under conditions that select for the continued presence of said selectable genetic marker, whereby recombination occurs between said third DNA segment and said fourth DNA segment, said recombination transferring said mutation to a specific site in said bacterial cell.
Claim 23
Dependent Claim
14 words
A gram-negative bacterial cell having a mutation made by the method of claim 21.
References Cited (Backward Citations)
No citation records indexed.
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1989-12-01 | 1990-06-02 | 1990-12-02 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1993-12-01 | 1994-06-02 | 1994-12-02 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1997-12-01 | 1998-06-02 | 1998-12-02 | $7700.00 | Window Closed / Lapsed |