Bacterial counterselectable hybrid systems

• US 7,504,216 B2
• Filed: 02/02/2005
• Issued: 03/17/2009
• Est. Priority Date: 02/02/2004
• Status: Expired due to Fees

• Current Assignee: University Of Massachusetts
• Original Assignee: University Of Massachusetts
• Inventors: Meng, Xiangdong, Wolfe, Scot Andrew, Joung, Jae Keith
• Primary Examiner(s): BURKHART, MICHAEL D
• Application Number:
  US11/050,174
• Publication Number:
  US 20050287550A1
• Time in Patent Office: 1,504 Days
• Field of Search: None
• US Class Current: 435/6.13
• CPC Class Codes: C12N 15/74 Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora

Abstract

The present application relates to the hybrid selection methods in prokaryotes using counterselectable reporter genes.

Claims

1. A method for determining whether a test DNA molecule interacts with a test polypeptide, the method comprising

(a) providing a first population of prokaryotic host cells, wherein at least one of the host cells comprises;

(i) a reporter vector comprising a selectable reporter gene and a counterselectable reporter gene, wherein the selectable reporter gene and the counterselectable reporter gene are both operably linked to a test DNA molecule; and

(ii) a chimeric gene that encodes a fusion protein comprising a test polypeptide fused to a gene activating domain, wherein interaction of the test DNA molecule and the test polypeptide in the host cell results in an increase in expression of the selectable reporter gene and of the counterselectable reporter gene;

(b) maintaining the first population of host cells under selective conditions that allow cell growth as a result of the expression of the selectable reporter gene;

(c) isolating the reporter vectors from cells of the first population of host cells that grow under the selective conditions;

(d) introducing the isolated reporter vectors into a second population of host cells that lack the chimeric gene;

(e) maintaining the second population of host cells under counterselective conditions that inhibit cell growth as a result of the expression of the counterselectable reporter gene; and

(f) measuring growth of the second population of host cells, wherein an ability to survive under counterselective conditions indicates that the test DNA molecule has interacted with the test polypeptide.

2. The method of claim 1, further comprising identifying a test DNA molecule that interacts with the test polypeptide, the method comprising isolating a reporter vector from a cell of the second population of host cells that grow under the counterselective conditions;

and determining the sequence of the test DNA molecule in the reporter vector.

3. The method of claim 1, wherein the test DNA molecule is from a nucleic acid library.

4. The method of claim 1, wherein the host cells lack a functional endogenous gene that is functionally homologous to the selectable reporter gene, and wherein the host cell is not capable of growing under selective conditions in the absence of the selectable reporter gene.

5. The method of claim 1, wherein the host cells lack a functional endogenous his-B gene.

6. The method of claim 1, wherein the host cells lack a functional endogenous gene that is functionally homologous to the counterselectable reporter gene, and wherein the host cell is capable of growing under counterselective conditions in the absence of the counterselectable reporter gene.

7. The method of claim 1, wherein the host cells lack a functional endogenous pyrF gene.

8. The method of claim 1, wherein the selectable reporter gene is selected from the group consisting of LEU2, TRP1, and HIS3.

9. The method of claim 8, wherein the selectable reporter gene is HIS3 and the selective conditions comprise maintaining the host cells in medium lacking histidine.

10. The method of claim 9, wherein the selective conditions further comprise maintaining the host cells in medium comprising 3-amino-1, 2, 4-triazole (3-AT).

11. The method of claim 1, wherein the counterselectable reporter gene is selected from the group consisting of URA3, LYS2, GAL1, CYH2, sacB, and CAN1.

12. The method of claim 11, wherein the counterselectable reporter gene is URA3 and the counterselective conditions comprise maintaining the host cells in medium comprising uracil and 5-fluoro orotic acid (5-FOA).

13. The method of claim 1, wherein the reporter gene encodes a gene product that provides at least one detectable signal selected from the group consisting of color, fluorescence, luminscence, a cell surface tag, cell viability, relief of a cell nutritional requirement, cell growth, and drug resistance.

14. The method of claim 1, wherein the reporter gene encodes a gene product selected from the group consisting of a spectinomycin resistance gene product, a streptomycin resistance gene, chloramphenicol acetyl transferase, luciferase, β

-galactosidase, and alkaline phosphatase.

15. The method of claim 1, wherein the host cell is selected from the group consisting of a strain of Escherichia, Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, and Shigella.

16. The method of claim 1, wherein the gene activating domain comprises the α

domain of RNA polymerase.

17. The method of claim 1, wherein the selectable reporter gene and the counterselectable reporter gene are operably linked to a lac promoter.