Difference between revisions of "Part:BBa K2986003"
| Line 8: | Line 8: | ||
[[File:The_structure_of_GVAPO.png ]] | [[File:The_structure_of_GVAPO.png ]] | ||
| − | + | Test their light-dependent impact on transcriptional activity of a firefly luciferase (Fluc) reporter driven by Gal4 binding sites upstream of a TATA box after transient transfection in HEK293 cells, illumination with 0.84 W m−2 460 nm peak light from an LED lamp for 22 h and measurement of expression. | |
| − | Test their light-dependent impact on transcriptional activity of a firefly luciferase (Fluc) reporter driven by Gal4 binding sites upstream of a TATA box after transient transfection in HEK293 cells, illumination with 0.84 W m−2 460 nm peak light from an LED lamp for 22 h and measurement of expression | + | |
[[File:Light-_dependent_activation_of_Fluc_reporters_based_on_GAVP_with_different_mutations_to_enhance_dimerization.png|400px|thumb|left|ali left]] | [[File:Light-_dependent_activation_of_Fluc_reporters_based_on_GAVP_with_different_mutations_to_enhance_dimerization.png|400px|thumb|left|ali left]] | ||
| − | + | Transactivators containing the p65 activation domain (GAVP) or the VP16 activation domain (GAVV) both showed marked light-induced reporter gene transcription, but the GAVP transactivator resulted in much greater gene expression under light exposure conditions. | |
Mutation of Cys108 in VVD to serine blocked light-inducible gene expression as expected13. | Mutation of Cys108 in VVD to serine blocked light-inducible gene expression as expected13. | ||
Mutation of Cys71 to valine in VVD is known to enhance the stability of the light-induced VVD dimer14, and based on the crystal structure of VVD13 we hypothesized that mutating Gln56 of VVD to lysine would form a salt bridge with Asp68 of the other VVD protein and additionaly stabilize the dimer. Both dimer-enhancing mutations, C71V and N56K, in the VVD domain decreased reporter gene expression in the dark, whereas the N56K,C71V double mutant (optimized GAVP (GAVPO)) additionally decreased the background gene expression to a minimal level. | Mutation of Cys71 to valine in VVD is known to enhance the stability of the light-induced VVD dimer14, and based on the crystal structure of VVD13 we hypothesized that mutating Gln56 of VVD to lysine would form a salt bridge with Asp68 of the other VVD protein and additionaly stabilize the dimer. Both dimer-enhancing mutations, C71V and N56K, in the VVD domain decreased reporter gene expression in the dark, whereas the N56K,C71V double mutant (optimized GAVP (GAVPO)) additionally decreased the background gene expression to a minimal level. | ||
Revision as of 03:11, 7 October 2019
light-switchable transactivator Basic part
Usage and Biology:
GVAPO is a synthetic light-switch transgene system, the transactivator binds promoters upon blue-light exposure and rapidly initiates transcription of target transgenes in mammalian cells and in mice. this transgene system provides a robust and convenient way to spatiotemporally control gene expression and can be used to manipulate many biological processes in living systems with minimal perturbation. Vivid (VVD), the smallest light-oxygen-voltage (LOV) domain–containing protein, forms a rapidly exchanging dimer upon blue-light activation. This part utilizes the DNA-binding property of a Gal4(65)-VVD fusion protein would be light-switchable, as light should induce dimerization of the fusion protein, enhance binding to the UASG sequence and activate transcription and removing the light should result in gradual dissociation of the dimers, DNA dissociation and inactivation.
Test their light-dependent impact on transcriptional activity of a firefly luciferase (Fluc) reporter driven by Gal4 binding sites upstream of a TATA box after transient transfection in HEK293 cells, illumination with 0.84 W m−2 460 nm peak light from an LED lamp for 22 h and measurement of expression.
Transactivators containing the p65 activation domain (GAVP) or the VP16 activation domain (GAVV) both showed marked light-induced reporter gene transcription, but the GAVP transactivator resulted in much greater gene expression under light exposure conditions. Mutation of Cys108 in VVD to serine blocked light-inducible gene expression as expected13. Mutation of Cys71 to valine in VVD is known to enhance the stability of the light-induced VVD dimer14, and based on the crystal structure of VVD13 we hypothesized that mutating Gln56 of VVD to lysine would form a salt bridge with Asp68 of the other VVD protein and additionaly stabilize the dimer. Both dimer-enhancing mutations, C71V and N56K, in the VVD domain decreased reporter gene expression in the dark, whereas the N56K,C71V double mutant (optimized GAVP (GAVPO)) additionally decreased the background gene expression to a minimal level. So We used GAVPO in all subsequent studies, and we referred to the gene promoter system based on GAVPO as the light-on (LightOn) system.
Design Notes
How can we coupled this light-switch system with gene coded for the cytokines, which may stay in the cytosol or secrete to the outside of the cells. Whether changing another kind of cells can affect the effect of this system.
Source
This light-switch transgene system is form the lab of Xue Wang, Xianjun Chen & Yi Yang, they published Spatiotemporal control of gene expression by a light-switchable transgene system in Nature Methods 2012.
SequenceAndFeatures
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
References
Wang X , Chen X , Yang Y . Spatiotemporal control of gene expression by a light-switchable transgene system[J]. Nature Methods, 2012, 9(3):266-269.