Universal light-switchable gene promoter system
View Patent ↗An artificial promoter system that can be fused upstream of any desired gene enabling reversible induction or repression of the expression of the gene at will in any suitable host cell or organisms by light is described. The design of the system is such that a molecule of the plant photoreceptor phytochrome is targeted to the specific DNA binding site in the promoter by a protein domain that is fused to the phytochrome and that specifically recognizes this binding site. This bound phytochrome, upon activation by light, recruits a second fusion protein consisting of a protein that binds to phytochrome only upon light activation and a transcriptional activation domain that activates expression of the gene downstream of the promoter.
1. A cell, comprising:
a target gene;
a first recombinant fusion protein including a phytochrome and a DNA binding protein and
a second recombinant fusion protein including a phytochrome interacting protein and a transcriptional regulatory protein.
2. The cell of claim 1 wherein the target gene is a recombinant target gene.
3. The cell of claim 1 wherein the target gene is a gene naturally occurring within said cell.
4. The cell of claim 1 further comprising one or more recombinant chromophore biosynthetic proteins.
5. The cell of claim 1 wherein said transcriptional regulatory protein is a repressor domain or an activator domain.
6. The cell of claim 1 further comprising an exogenously supplied chromophore.
7. The cell of claim 1 wherein said cell is selected from the group consisting of plant, algae, fungi, yeast and animal cells.
8. The cell of claim 7 wherein said animal cell is selected from the group consisting of mammalian, insect, worm, and fish cells.
9. The cell of claim 8 wherein said mammalian cell is an isolated human cell.
10. The cell of claim 1 wherein said transcriptional regulatory protein is an activator.
11. The cell of claim 1 wherein said transcriptional regulatory protein is a repressor.
12. A cell, comprising:
a target gene;
a first recombinant DNA construct encoding a fusion protein including a phytochrome and a DNA binding protein and
a second recombinant DNA construct encoding a fusion protein including a phytochrome interacting protein and a transcriptional regulatory protein.
13. The cell of claim 12 wherein said target gene is a recombinant target gene.
14. The cell of claim 12 wherein the target gene is a gene naturally occurring within said cell.
15. The cell of claim 12 wherein said first and said second DNA constructs further include a promoter active in said cell.
16. The cell of claim 12 further comprising a third DNA construct encoding a chromophore biosynthetic protein.
17. A kit, comprising:
a first DNA construct encoding a fusion protein including a phytochrome and a DNA binding protein and
a second DNA construct encoding a fusion protein including a phytochrome interacting protein and a transcriptional regulatory protein.
18. The kit of claim 17 wherein said DNA constructs are cloned into one or more vectors.
19. The kit of claim 17 further including a third DNA construct encoding a target gene.
20. The kit of claim 17 further including instructions for use.
21. The kit of claim 17 further including a light source.
22. The kit of claim 17 further including a chromophore.
23. The kit of claim 17 further including a DNA construct encoding a chromophore biosynthetic protein.
24. The kit of claim 17 wherein said transcriptional regulatory protein is an activator.
25. The kit of claim 17 wherein said transcriptional regulatory protein is a repressor.
26. A method of regulating the expression of a target gene by light, comprising:
a) incubating a cell, said cell comprising:
i) said target gene wherein said target gene has a promoter;
ii) a phytochrome chromophore;
iii) a first recombinant fusion protein including a phytochrome with Pr and Pfr conformers and a DNA binding protein that binds to said promoter; and
iv) a second recombinant fusion protein including a phytochrome interacting protein and a transcriptional regulatory protein
in the dark, under conditions so that the phytochrome chromophore attaches to the phytochrome to generate said Pr conformer and the DNA binding protein binds to its DNA binding site in said promoter, and
b) exposing said cell to sufficient light to convert the Pr conformer to the Pfr conformer to thereby permit the phytochrome to bind to the phytochrome interacting protein to thereby permit the second DNA binding protein protein to regulate the expression of the target gene.
27. The method of claim 26 wherein said transcriptional regulatory protein is an activator.
28. The method of claim 26 wherein said transcriptional regulatory protein is a repressor.
29. The method of claim 26 wherein said target gene is a recombinant target gene.
30. The method of claim 26 wherein said cell is selected from the group consisting of plant, algae, fungi, yeast and animal cells.
31. The method of claim 30 wherein said animal cell is a mammalian cell.
32. The method of claim 31 wherein said mammalian cell is an isolated human cell.
33. The method of claim 26 wherein said chromophore is exogenously supplied.