Patent FR1230487A successfully refreshed from Google Patents.
US4743546A
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Controlled gene excision
Assignee: BTI AND HJ HEINZ COMPANY A CORP OF PA
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Filing: 1985-02-13
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Granted: 1988-05-10
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Expires: 2005-02-13
Abstract
Abstract An Escherichia coli chromosomal DNA segment is engineered for controllable excision and loss from the E. coli cell population. The DNA segment has a gene determining a function that is lost in the absence of that chromosomal segment. Specifically, the segment includes a pair of lambdoid phage att sites positioned at opposite ends thereof, and a gene encoding the function. The E. coli cell includes DNA encoding lambdoid phage int and xis, positioned for transcription under control of an external stimulus. By excising the chromosomal DNA segment once a desired cell population is achieved, cell growth and diversion of intermediates from a desired fermentation product are diminished or avoided.
Inventors
- 👤 Keith C. Backman
- 👤 Ramaswamy Balakrishnan
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-09 08:33
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/67; C12N15/11; C12N15/52; C12N15/87; C12N15/90; C12P; C12P13/00; C12P13/04; C12P13/22; Y; Y10; Y10S; Y10S435/00; Y10S435/8215; Y10S435/822; Y10S435/848; Y10S435/849
Claims Analysis
Total Claims: 32
Claim 1
Independent Claim
117 words
An Escherichia coli bacterial cell which undergoes controllable excision of a genetically engineered, chromosomally located DNA segment, wherein said cell comprises: DNA encoding lambdoid int and xis genes, operably positioned to be transcribed under the control of an external stimulus, wherein said external stimulus causes excision of a chromosomally located DNA segment, and a gene function is lost thereby from said cell and wherein said chromosomally located DNA segment, when excised, is incapable of replication, and wherein said DNA segment comprises a pair of lambdoid phage att sites, positioned at opposite ends thereof and a gene determining a function in said cell, and wherein said function is lost in the absence of said chromosomally located DNA segment.
Claim 2
Dependent Claim
117 words
An Escherichia coli bacterial cell which undergoes controllable excision of a genetically engineered, chromosomally located DNA segment, wherein said cell comprises: DNA encoding lambdoid int and xis genes, operably positioned to be transcribed under the control of an external stimulus, wherein said external stimulus causes excision of a chromosomally located DNA segment, and a gene function is lost thereby from said cell and wherein said chromosomally located DNA segment, when excised, is incapable of replication, and wherein said DNA segment comprises a pair of lambdoid phage att sites, positioned at opposite ends thereof and a gene determining a function in said cell, and wherein said function is lost in the absence of said chromosomally located DNA segment.
Claim 3
Dependent Claim
34 words
The E. coli cell of claim 1 wherein said genetically engineered, chromosomally located DNA segment lacks sufficient homology to integrate into said chromosome in the absence of expression of a lambdoid phage int gene.
Claim 4
Dependent Claim
29 words
The E. coli cell of claim 1 wherein said DNA encoding int and xis genes are operably positioned for transcription under the control of lambdoid phage cI gene product.
Claim 5
Dependent Claim
27 words
The E. coli cell of claim 1 wherein said DNA encoding int and xis genes are operably positioned on said chromosomally located DNA segment to be excised.
Claim 6
Dependent Claim
57 words
The E. coli cell of claim 1 wherein said cell further comprises a helper phage comprising a lambdoid phage int gene, and wherein said helper phage effects integration of said chromosomally located DNA segment into the chromosome of said cell prior to the controlled excision of said segment and subsequent loss of said segment from said cell.
Claim 7
Dependent Claim
20 words
The E. coli cell of claim 1 wherein said gene function is part of an aromatic amino acid synthesis pathway.
Claim 8
Dependent Claim
15 words
The E. coli cell of claim 6, wherein said gene is tyrA, tyrR, or trpE.
Claim 9
Dependent Claim
16 words
The E. coli cell of claim 6, wherein said gene is trpR, aroP, tyrA, or pheA.
Claim 10
Dependent Claim
19 words
The E. coli cell of claim 1, wherein said gene codes for an enzyme in the isoleucine biosynthesis pathway.
Claim 11
Dependent Claim
14 words
The E. coli cell of claim 1, wherein said gene codes for proline oxidase.
Claim 12
Dependent Claim
12 words
The E. coli cell of claim 1, wherein said gene is ppc.
Claim 13
Dependent Claim
66 words
The E. coli cell of claim 1, wherein said chromosomally located DNA segment comprises DNA from a plasmid that has been integrated in said cell, wherein said plasmid is: a derivative of pKB800, deposited as ATCC 53019, wherein ori has been deleted and into which derivative said gene has been inserted; or a derivative of pKB803, deposited as ATCC 53020, from which ori has been deleted.
Claim 14
Dependent Claim
62 words
A method of producing a desired compound comprising: culturing the E. coli cells of claim 1 to a desired population density; controlling said external stimulus that causes excision of said gene from the chromosomes of members of said E. coli cell population; culturing cells in a medium wherein said cells have said gene excised from their chromosomes; recovering said compound produced thereby.
Claim 15
Dependent Claim
27 words
The method of claim 13 wherein said gene codes for a function and said medium lacks components required to permit growth in the absence of said function.
Claim 16
Dependent Claim
46 words
The method of claim 13 or claim 14 wherein said E. coli cells produce said desired compound via a synthesis pathway comprising intermediate compounds, and wherein said gene codes for an enzyme that converts an intermediate of said pathway to a compound outside said synthesis pathway.
Claim 17
Dependent Claim
46 words
The method of claim 13 wherein said E. coli cells produce said desired compound via a synthesis pathway comprising intermediate compounds, wherein said gene codes for a function regulating expression of an enzyme, and wherein one of said intermediate compounds is a substrate for said enzyme.
Claim 18
Dependent Claim
30 words
The method of claim 15, wherein said desired compound is phenylalanine and wherein said gene codes for an enzyme that catalyzes a step in the synthesis of tyrosine or tryptophan.
Claim 19
Dependent Claim
26 words
The method of claim 13, wherein said desired compound is phenylalanine, and wherein said gene is selected from the group consisting of tyrA, tyrR and trpE.
Claim 20
Dependent Claim
27 words
The method of claim 13, wherein said desired compound is tryptophan and said gene is selected from the group consisting of tna, trR, aroP, tyrA, or pheA.
Claim 21
Dependent Claim
23 words
The method of claim 14, wherein said compound is threonine and wherein said gene codes for an enzyme in the isoleucine biosynthesis pathway.
Claim 22
Dependent Claim
18 words
The method of claim 14, wherein said compound is proline and wherein said gene codes for proline oxidase.
Claim 23
Dependent Claim
10 words
The method of claim 13, wherein said gene is ppc.
Claim 24
Dependent Claim
92 words
A method of making the E. coli cell of claim 1, wherein the loss of expresssion of said gene is controlled, said method comprising: (a) transforming a host cell with an engineered plasmid, wherein said plasmid comprises a DNA segment that lacks the ability to extrachromosomally replicate in said cell, and wherein said DNA segment comprises said gene and a lambdoid phage att site; (b) allowing integration and expression of said gene in said E. coli cell; and then (c) effecting the excision of said chromosomal DNA segment by said extenal stimulus.
Claim 25
Dependent Claim
61 words
An engineered plasmid that lacks the ability to replicate in E. coli wherein said plasmid comprises: a lambdoid att site; a gene encoding a function whose expression is to be controlled; and int and xis genes from a lambdoid phage, wherein said int and xis genes are operably positioned and oriented for expression under the control of a lambdoid cI gene.
Claim 26
Dependent Claim
61 words
An engineered plasmid that lacks the ability to replicate in E. coli wherein said plasmid comprises: a lambdoid att site; a gene encoding a function whose expression is to be controlled; and int and xis genes from a lambdoid phage, wherein said int and xis genes are operably positioned and oriented for expression under the control of a lambdoid cI gene.
Claim 27
Dependent Claim
40 words
The plasmid of claim 24 wherein said plasmid is: a derivative of pKB800 ATCC 53019 wherein ori has been deleted and into which said gene has been inserted or a derivative of pKB803 ATCC 53020 wherein ori has been deleted.
Claim 28
Dependent Claim
13 words
The plasmid of claim 24 wherein said gene is tyrA, tyrR, or trpE.
Claim 29
Dependent Claim
13 words
The plasmid of claim 24, wherein gene is trpR, aroP, tyrA, or pheA.
Claim 30
Dependent Claim
17 words
The plasmid of claim 24, wherein said gene codes for an enzyme in the isoleucine biosynthesis pathway.
Claim 31
Dependent Claim
12 words
The plasmid of claim 24, wherein said gene codes for proline oxidase.
Claim 32
Dependent Claim
10 words
The plasmid of claim 24, wherein said gene is ppc.
References Cited (Backward Citations)
No citation records indexed.
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