Phytochrome-based fluorophores
Genetically-engineered fluorophore molecules with increased fluorescence are provided. These fluorophores are derived from the domains of phytochromes, and in particular bacterial phytochromes. Methods for generating these fluorophores and various applications of these fluorophores are also provided.
1. A cell that comprises a DNA molecule having a regulatory element from a gene, other than a gene encoding a Deinococcus radiodurans phytochrome, operably linked to a DNA sequence that encodes a polypeptide that is (i) at least 80% identical to the chromophore-binding domain of SEQ ID NO: 1; or (ii) at least 80% identical to the sequence of SEQ ID NO: 2; wherein the polypeptide has increased fluorescent activity over the corresponding wild-type phytochrome domain, wherein the polypeptide comprises a D207H mutation from the phytochrome of Deinococcus radiodurans.
2. The cell of claim 1 , wherein the cell is selected from the group consisting of a bacterial cell, a yeast cell, a fungal cell, a plant cell, an insect cell, a nematode cell, an animal cell, and a human cell.
3. An isolated polynucleotide comprising a sequence that encodes a polypeptide that is (i) at least 80% identical to the chromophore-binding domain of SEQ ID NO: 1; or (ii) at least 80% identical to the sequence of SEQ ID NO: 2; wherein the polypeptide contains a His rather than an Asp in the Deinococcus radiodurans DIP motif, wherein the polypeptide has increased fluorescent activity over the corresponding wild-type phytochrome domain.
4. The polynucleotide of claim 3 , wherein the sequence encodes a polypeptide that is at least 80% identical to SEQ ID NO: 2.
5. The polynucleotide of claim 4 , wherein the sequence encodes a polypeptide that is at least 95% identical to the chromophore binding domain of SEQ ID NO: 1, at least 95% identical to SEQ ID NO: 2, or a combination thereof.
6. The polynucleotide of claim 4 , wherein the increased fluorescent activity comprises an intensity that is at least 75% greater compared with the corresponding wild-type domain.
7. The polynucleotide of claim 3 , wherein the increased fluorescent activity comprises an intensity that is at least 75% greater compared with the corresponding wild-type domain.
8. The polynucleotide of claim 3 , wherein the increased fluorescent activity comprises an intensity that is at least 200% greater compared with the corresponding wild-type domain.
9. The polynucleotide of claim 3 , wherein the polypeptide comprises an Ala rather than an Ile in the Deinococcus radiodurans DIP motif.
10. The polynucleotide of claim 3 , comprising the sequence encoding a modified phytochrome domain having increased fluorescent activity shifted to the far red.
11. An expression vector comprising the isolated polynucleotide of claim 3 and a regulatory sequence operably linked to the polynucleotide.
12. A cell comprising the polynucleotide of claim 3 .
13. The cell of claim 12 , wherein the cell is selected from the group consisting of a bacterial cell, a fungal cell, an insect cell, a nematode cell and an animal cell.
14. The cell of claim 13 , wherein the cell is a human cell or a yeast cell.
15. The cell of claim 1 , wherein the polypeptide further comprises one or more mutations of the phytochrome of Deinococcus radiodurans selected from E25A, E27A, I35A, P37A, Y176H, F203A, F203H, F203W, I208A, Y216H, Y216W, R254A, R254K, Y263H, H290N and H290Q.
16. An isolated polynucleotide comprising a sequence that encodes a polypeptide that is (i) at least 80% identical to the chromophore-binding domain of SEQ ID NO: 1; or (ii) at least 80% identical to SEQ ID NO: 2; and with increased fluorescent activity over the corresponding wild-type phytochrome domain, wherein the polypeptide comprises a D207H mutation from the phytochrome of Deinococcus radiodurans.
17. The polynucleotide of claim 16 , wherein the polypeptide further comprises one or more mutations of the phytochrome of Deinococcus radiodurans selected from E25A, E27A, I35A, P37A, Y176H, F203A, F203H, F203W, I208A, Y216H, Y216W, R254A, R254K, Y263H, H290N and H290Q.