Oxygen-responsive bacterial gene switch
The invention provided herein relates to sequence determinants that elicit certain levels of gene expression and methods for obtaining engineered ligand-responsive gene switches comprising these sequence determinants. More particularly, the invention provided herein relates to molecular building blocks (i.e., discrete nucleotide sequences), synthetic ligand-responsive gene switches comprising an assembly of these molecular building blocks, and methods of using synthetic ligand-responsive gene switches as customizable and controllable expression systems and sensors.
1. A vector comprising a synthetic gene switch comprising
(i) a first sequence determinant portion selected from the group consisting of SEQ ID NOs:2, 4, 36, 37, 38, and 39;
(ii) a second sequence determinant portion selected from the group consisting of SEQ ID NOs:2, 4, 36, 37, 38, and 39; and
(iii) a third sequence determinant portion selected from the group consisting of SEQ ID NOs:2, 4, 36, 37, 38, and 39,
wherein the first and second sequence determinant portions are separated by an 11-basepair center-to-center spacing interval, the second and third sequence determinant portions are separated by an 11-basepair center-to-center spacing interval, and the first and third sequence determinant portions are separated by a 22-basepair center-to-center spacing interval, and with the proviso that if the first sequence determinant portion is SEQ ID NO:2, the second sequence determinant portion is SEQ ID NO:37, and the third sequence determinant portion is SEQ ID NO:39 the synthetic gene switch additionally comprises a fourth sequence determinant portion selected from the group consisting of SEQ ID NOs:2, 4, 36, 37, 38, and 39,
wherein the synthetic gene switch is operably linked to a promoter.
2. The vector of claim 1 , wherein the synthetic gene switch is positioned with respect to the promoter on the vector to confer ArcA-dependent promoter activity.
3. A bacterial host cell comprising the vector of claim 1 .
4. The bacterial host cell of claim 3 , wherein the cell is an E. coli cell.
5. The vector of claim 1 , wherein the synthetic gene switch comprises a sequence selected from the group consisting of SEQ ID NOs:5-16.
6. The vector of claim 5 , wherein the synthetic gene switch comprises a sequence selected from the group consisting of SEQ ID NOs:18-32.
7. An expression vector comprising,
a synthetic gene switch comprising:
(i) a first sequence determinant portion selected from the group consisting of SEQ ID NOs:2, 4, 36, 37, 38, and 39;
(ii) a second sequence determinant portion selected from the group consisting of SEQ ID NOs:2, 4, 36, 37, 38, and 39; and
(iii) a third sequence determinant portion selected from the group consisting of SEQ ID NOs:2, 4, 36, 37, 38, and 39,
wherein the first and second sequence determinant portions are separated by an 11-basepair center-to-center spacing interval, the second and third sequence determinant portions are separated by an 11-basepair center-to-center spacing interval, and the first and third sequence determinant portions are separated by a 22-basepair center-to-center spacing interval, and with the proviso that if the first sequence determinant portion is SEQ ID NO:2, the second sequence determinant portion is SEQ ID NO:37, and the third sequence determinant portion is SEQ ID NO:39, the synthetic gene switch additionally comprises a fourth sequence determinant portion selected from the group consisting of SEQ ID NOs:2, 4, 36, 37, 38, and 39,
wherein the synthetic gene switch additionally comprises at least a portion of an RNA polymerase binding site; and
a target nucleic acid sequence downstream of the synthetic gene switch.
8. The expression vector of claim 7 , wherein the synthetic gene switch is operably linked to a promoter.
9. A bacterial host cell comprising the vector of claim 7 .
10. The bacterial host cell of claim 9 , wherein the cell is an E. coli cell.
11. The expression vector of claim 7 , wherein the synthetic gene switch comprises a fourth sequence determinant portion selected from the group consisting of SEQ ID NOs:2, 4, 36, 37, 38, and 39 and wherein the fourth sequence determinant portion is separated from either the first or third sequence determinant portions by an 11-basepair center-to-center spacing interval.
12. The expression vector of claim 7 , wherein the RNA polymerase binding site is a binding site for bacterial σ 70 -RNA polymerase.
13. The vector of claim 7 , wherein the synthetic gene switch comprises a sequence selected from the group consisting of SEQ ID NOs:5-16.
14. The vector of claim 13 , wherein the synthetic gene switch comprises a sequence selected from the group consisting of SEQ ID NOs:18-32.
15. A method for modulating gene expression of a target nucleic acid sequence, the method comprising:
introducing into a host cell an expression vector of claim 7 ; and
subjecting the host cell to a change in oxygen concentration until the gene expression level of the target nucleic acid sequence is modulated.
16. The method of claim 15 , wherein the gene expression level is modulated under anaerobic conditions.
17. An expression vector comprising,
a synthetic gene switch comprising:
(i) a first sequence determinant portion selected from the group consisting of SEQ ID NOs:2, 4, 36, 37, 38, and 39;
(ii) a second sequence determinant portion selected from the group consisting of SEQ ID NOs:2, 4, 36, 37, 38, and 39; and
(iii) a third sequence determinant portion selected from the group consisting of SEQ ID NOs:2, 4, 36, 37, 38, and 39,
wherein the first and second sequence determinant portions are separated by an 11-basepair center-to-center spacing interval, the second and third sequence determinant portions are separated by an 11-basepair center-to-center spacing interval, and the first and third sequence determinant portions are separated by a 22-basepair center-to-center spacing interval, and with the proviso that if the first sequence determinant portion is SEQ ID NO:2, the second sequence determinant portion is SEQ ID NO:37, and the third sequence determinant portion is SEQ ID NO:39, the synthetic gene switch additionally comprises a fourth sequence determinant portion selected from the group consisting of SEQ ID NOs:2, 4, 36, 37, 38, and 39,
wherein the synthetic gene switch comprises a promoter element; and
a target nucleic acid sequence downstream of the synthetic gene switch.
18. The vector of claim 17 , wherein the synthetic gene switch comprises a sequence selected from the group consisting of SEQ ID NOs:5-16.
19. The vector of claim 18 , wherein the synthetic gene switch comprises a sequence selected from the group consisting of SEQ ID NOs:18-32.
20. A bacterial host cell comprising the vector of claim 17 .
21. The bacterial host cell of claim 20 , wherein the cell is an E. coli cell.