Precisely regulated expression of deleterious genes

• US 5,196,318 A
• Filed: 06/26/1990
• Issued: 03/23/1993
• Est. Priority Date: 06/26/1990
• Status: Expired due to Term

• Current Assignee: Texas A&M University System
• Original Assignee: Texas A&M University System
• Inventors: Baldwin, Thomas O., Shadel, Gerald S., Devine, Jerry H.
• Primary Examiner(s): Schwartz, Richard A.
• Assistant Examiner(s): CARTER, PHILIP
• Application Number:
  US07/544,268
• Time in Patent Office: 1,001 Days
• Field of Search: 435/320.1, 435/172.3, 435/69.1, 536/27
• US Class Current: 435/69.1
• CPC Class Codes: C07K 14/28 from Vibrionaceae (F)
• CPC Class Codes: C12N 15/635 Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli

Abstract

The invention relates to an expression vector system based on the regulation of bacterial luminescence (the lux gene system). The invention further relates to the construction of a precisely regulatable expression vector system which comprises a complete luxR gene in combination with an inactivated luxI gene. If the system is turned off, no significant transcription occurs of any cloned gene product when used in combination with the regulatory scheme of the invention as is demonstrated by using the bacteriophage λ lysis genes. The induction of transcription relies on the addition of exogenous autoinducer which is both inexpensive and easy-to-use and which is required in only minute amounts.

Claims

1. An expression system comprising a lux regulatory circuit, said regulatory circuit further comprising a luxR gene and truncated luxI gene of Vibrio, operably linked to and capable of stringently regulating expression of a gene, a product of which said gene is deleterious to an Escherichia coil host cell in which said expression system and said gene are resident.

2. The expression system of claim 1 wherein the lux regulatory circuit further comprises a luxR gene derived from the leftward operon of said lux regulatory circuit, said luxR being operably linked to a control region of said lux regulatory circuit, said control region being operably linked to a portion of a rightward operon, said rightward operon retaining a truncated luxI gene.

3. The expression system of claim 1 wherein said deleterious gene product is the product of a gene which, when expressed, is lethal to said host cell in which said expression system and said gene are resident.

4. The expression system of claim 3 wherein said gene is a lysis gene of bacteriophage λ

.

5. The expression system of claim 1 wherein induction of said expression is controlled by addition of an inducer to the environment of said host cells in which said expression system is resident.

6. The expression of claim 5 wherein said inducer is an exogenously added inducer.

7. The expression of claim 5 wherein said inducer is N-(3-oxo-hexanoyl) homoserine lactone.

8. The expression of claim 1 wherein said lux regulatory circuit is operably linked to and capable of exerting control over at least one secondary regulatory system.

9. The expression system of claim 1 wherein the lux regulatory circuit further comprises a luxR gene derived from the leftward operon of said lux regulatory circuit, said luxR gene being operably linked to a control region of said lux regulatory circuit, said control region being operably linked to a portion of a rightward operon, said rightward operon comprising a promoter and a truncated luxI gene.

10. A vector comprising a lux regulatory circuit, said regulatory circuit further comprising a luxR gene and truncated luxI gene of Vibrio, operably linked to and capable of stringently regulating expression of a gene, a product of which said gene is deleterious to an Escherichia coli host cell in which said lux regulatory circuit and said gene are resident.

11. The vector of claim 10 wherein the lux regulatory circuit further comprises a luxR gene derived from the leftward operon of said lux regulatory circuit, said luxR being operably linked to a control region of said lux regulatory circuit, said control region being operably linked to a portion of a rightward operon, said rightward operon retaining a truncated luxI gene.

12. The vector of claim 11 wherein said truncated luxI gene is operably linked downstream of its truncated portion to a multiple cloning site.

13. The vector of claim 11 wherein the vector is pGS103.

14. A method for expression of a deleterious gene comprising:

(a) constructing a vector which is comprised of a lux regulatory circuit, said regulatory circuit further comprising a luxR gene and truncated luxI gene of Vibrio, capable of being operably linked to and capable of stringently regulating expression of said deleterious gene;

(b) operably linking said deleterious gene to said lux regulatory circuit;

(c) inserting said vector containing said lux regulatory circuit operably linked to said deleterious gene into a suitable Escherichia coli host cell;

(d) allowing multiplication and growth of said host cell to a suitable level; and

,

(e) inducing expression of said deleterious gene by introduction of an exogenous inducer into the environment of said host cell.

15. The method of claim 14 wherein said deleterious gene produces a product which, when expressed, is lethal to said host cell.