Process for producing a cloned luciferase-synthesizing microorganism

• US 4,581,335 A
• Filed: 12/01/1982
• Issued: 04/08/1986
• Est. Priority Date: 12/01/1982
• Status: Expired due to Term

• Current Assignee: Texas A&M University System
• Original Assignee: Texas A&M University System
• Inventors: Baldwin, Thomas O.
• Primary Examiner(s): NOT, DEFINED
• Application Number:
  US06/445,697
• Time in Patent Office: 1,224 Days
• Field of Search: 435/172.3, 435/253, 435/172, 435/317, 435/189, 935/27
• US Class Current: 435/6.13
• CPC Class Codes: C12N 9/0071 acting on paired donors with incorporation of molecular oxygen (1.14)
• CPC Class Codes: C12Y 114/14003 Alkanal monooxygenase FMN (1.14.14.3), i.e. bacterial-luciferase
• CPC Class Codes: Y10S 435/849 Escherichia coli

Abstract

A process for producing a cloned luciferase-synthesizing microorganism, capable of the expression of bioluminescence, is disclosed. The process involves isolation and digestion of the genomic DNA of an appropriate bioluminescent microorganism, such as Vibrio harveyi, to obtain a DNA fragment which codes for luciferase. The DNA fragment is inserted into a pre-existing cloning vehicle under the control of an expression promoter, thereby producing a recombinant or derivatized cloning vehicle. A host microorganism is transformed with the derivatized cloning vehicle, cultured and purified for the expression of bioluminescence.

Claims

1. A process for producing a cloned luciferase-synthesizing microorganism capable of expressing bioluminescence comprising:

isolating genomic DNA of Vibrio harveyi;

digesting said genomic DNA to obtain a DNA fragment comprising an approximate 4.0 kilobase pair restriction fragment encoding for both the alpha and beta subunits of luciferase-synthesizing gene;

inserting said DNA fragment into a DNA cloning vehicle able to replicate in a host microorganism proximate to a promoter thereby producing a derivatized cloning vehicle;

transforming a host microorganism with said derivatized cloning vehicle to produce a transformed microorganism which expresses bioluminescence;

culturing said transformed microorganism; and

purifying said transformed microorganism for the expression of bioluminescence.

2. The process of claim 1 wherein said DNA cloning vehicle is plasmid DNA.

3. The process in claim 1 wherein said DNA cloning vehicle is pBR322.

4. The process in claim 1 wherein said derivatized cloning vehicle is pTB7.

5. The process in claim 1 wherein said DNA fragment is approximately a 4.0 kilobase pair Hind III fragment.

6. The process in claim 1 wherein said DNA fragment is approximately a 4.0 kilobase pair fragment selected from one of SalI-EcoRI;

SalI-Hind III;

SalI-BamHI;

BamHI-EcoRI;

BamHI-Hind III;

BamHI-BamHI;

Hind III-EcoRI;

or Hind III-BamHI.

7. A cloning vehicle having a DNA sequence which is able to replicate in a host microorganism, said cloning vehicle comprising:

a DNA fragment from Vibrio harveyi comprising approximately 4.0 kilobase pair restriction fragment encoding for both the alpha and beta subunits of luciferase-synthesizing gene; and

an expression promoter in operable proximity to said DNA fragment.

8. The cloning vehicle of claim 7 wherein said DNA sequence is plasmid DNA.

9. The cloning vehicle of claim 7 wherein said DNA fragment is approximately a 4.0 kilobase pair Hind III fragment.

10. The cloning vehicle of claim 7 wherein said DNA fragment is approximately a 4.0 kilobase pair selected from SalI-EcoRI;

SalI-Hind III;

SalI-BamHI;

BamHI-EcoRI;

BamHI-Hind III;

BamHI-BamHI;

Hind III-EcoRI;

or Hind III-BamHI.

11. The cloning vehicle of claim 8, wherein said cloning vehicle is derived from pBR322.

12. The cloning vehicle of claim 11 wherein the promoter is located in the tetracycline resistance region of pBR322.

13. The cloning vehicle of claim 8, wherein said cloning vehicle is pTB7.

14. A microorganism transformed by a cloning vehicle of claims 7, 8, 9, 10, 11, 12, or 13.

15. A microorganism transformed by a cloning vehicle of claims 7, 8, 9, 10, 11, 12, or 13 wherein expression of said DNA fragment produces bioluminescence upon addition of an aldehyde.

16. A transformed microorganism E. coli RR1/pTB7.