US4725535A
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Promoter probe vectors
Assignee: Tufts University
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Filing: 1983-02-22
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Granted: 1988-02-16
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Expires: 2003-02-22
Abstract
Abstract Promoter probe vectors for determining the presence of efficient promoter regions on DNA segments, which segments permit microbiological expression of genetic information. The promoter probe vector comprises replicons active in E. coli and B. subtilis, a structural gene for B-galactosidase and at least one endonuclease restriction site suitable for insertion of promoter-containing DNA fragments.
Inventors
- 👤 Abraham L. Sonenshein
- 👤 Caroline E. Donnelly
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-04 11:21
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/74; C12N15/75; C12N15/70; C12Q; C12Q1/00; C12Q1/68; C12Q1/6897
Claims Analysis
Total Claims: 17
Claim 1
Independent Claim
37 words
A promoter probe vector, comprising a replicon active in Escherichia coli, a replicon active in Bacillus subtilis, a structural gene for B-galactosidase, and at least one endonuclease restriction site suitable for insertion of promoter-containing fragments of DNA.
Claim 2
Dependent Claim
37 words
A promoter probe vector, comprising a replicon active in Escherichia coli, a replicon active in Bacillus subtilis, a structural gene for B-galactosidase, and at least one endonuclease restriction site suitable for insertion of promoter-containing fragments of DNA.
Claim 3
Dependent Claim
43 words
The vector of claim 1, wherein the replicon active in Escherichia coli comprises a replicon obtained directly or indirectly from plasmid pBR 322, and wherein the vector is useable to identify promoter sequences which are active in both Escherichia coli and Bacillus subtilis.
Claim 4
Dependent Claim
22 words
The vector of claim 1, wherein the replicon active in Bacillus subtilis comprises a replicon obtained directly or indirectly from plasmid pUB110.
Claim 5
Dependent Claim
18 words
The vector of claim 1, wherein the structural gene for B-galactosidase is the lacZ gene from Escherichia coli.
Claim 6
Dependent Claim
32 words
The vector of claim 1, wherein the endonuclease restriction site is a cleavage site for at least restriction endonuclease selected from the group of AvaI, BamHI, Bg1II, HindIII, PstI, EcoR1 and SalI.
Claim 7
Dependent Claim
12 words
The vector of claim 1, said vector having the properties of pCED6.
Claim 8
Dependent Claim
15 words
The vector of claim 6, said vector being pCED6, or a derivative or mutant thereof.
Claim 9
Dependent Claim
20 words
The vector of claim 1, said vector further carrying the structural gene for at least one type of antibiotic resistance.
Claim 10
Independent Claim
66 words
A method of testing DNA segments for the presence of a promoter, comprising inserting the DNA segments into a promoter probe vector to form a test plasmid, which vector comprises a replicon for Escherichia coli, a replicon for Bacillus subtilis and a structural gene for B-galactosidase, transforming Escherichia coli or Bacillus subtilis with the test plasmid, and analyzing the products made by the transformant for B-galactosidase.
Claim 11
Independent Claim
66 words
A method of testing DNA segments for the presence of a promoter, comprising inserting the DNA segments into a promoter probe vector to form a test plasmid, which vector comprises a replicon for Escherichia coli, a replicon for Bacillus subtilis and a structural gene for B-galactosidase, transforming Escherichia coli or Bacillus subtilis with the test plasmid, and analyzing the products made by the transformant for B-galactosidase.
Claim 12
Dependent Claim
58 words
The method of claim 9, wherein Escherichia coli is first transformed with the promoter probe vector containing the promoter, and then Bacillus subtilis is transformed with the promoter probe vector containing the promoter, and the analysis for B-galactosidase is carried out by contacting the bacteria with a substance which undergoes a color change in the presence of B-galactosidase.
Claim 13
Dependent Claim
17 words
The method of claim 10, wherein the substance used to analyze for B-galactosidase is 5-bromo-4-chloro-3-inodolyl-B-D-galactoside, or o-nitrophenyl-B-D-galactoside.
Claim 14
Independent Claim
157 words
A method of constructing a plasmid for use in expression of a product by Bacillus subtilis, said product being encoded by a product gene, comprising: (a) identifying a promoter useful for promoting expression of genetic products by Bacillus subtilis by (1) inserting promoter DNA segments into a promoter probe vector to form a test vector, said probe vector comprising a replicon for Escherichia coli, a replicon from Bacillus subtilis and a structural gene for B galactosidase, (2) transforming Escherichia coli or Bacillus subtilis with said test vector to form at least one transformant, and (3) recovering the promoter-containing test vector from at least one transformant which produced B-galactosidase; (b) forming a production vector by inserting a structural gene which codes for the desired product in the test vector; (c) transforming Bacillus subtilis with the production vector; (d) incubating the transformed Bacillus subtilis under conditions which permit production of the desired product, and (e) recovering the desired product.
Claim 15
Independent Claim
157 words
A method of constructing a plasmid for use in expression of a product by Bacillus subtilis, said product being encoded by a product gene, comprising: (a) identifying a promoter useful for promoting expression of genetic products by Bacillus subtilis by (1) inserting promoter DNA segments into a promoter probe vector to form a test vector, said probe vector comprising a replicon for Escherichia coli, a replicon from Bacillus subtilis and a structural gene for B galactosidase, (2) transforming Escherichia coli or Bacillus subtilis with said test vector to form at least one transformant, and (3) recovering the promoter-containing test vector from at least one transformant which produced B-galactosidase; (b) forming a production vector by inserting a structural gene which codes for the desired product in the test vector; (c) transforming Bacillus subtilis with the production vector; (d) incubating the transformed Bacillus subtilis under conditions which permit production of the desired product, and (e) recovering the desired product.
Claim 16
Dependent Claim
21 words
The method of claim 12, wherein the probe vector is a plasmid having the properties of pCED6 or a derivative thereof.
Claim 17
Dependent Claim
20 words
The method of claim 13, wherein the probe vector is the production vector produced by the method of claim 12.
References Cited (Backward Citations)
No citation records indexed.
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