Part:BBa_K1150026
uniCAS Red Light Switch Part II - Activator
| CMV:PhyB-Linker-VP16-NLS:BGH | |
|---|---|
| Function | Activation domain of red light
induced gene expression control |
| Use in | Mammalian cells |
| RFC standard | RFC 25 |
| Backbone | pSB1C3 |
| Submitted by | [http://2013.igem.org/Team:Freiburg Freiburg 2013] |
Usage and Biology
Fusion protein of PhyB and VP16. This fusion protein PhyB-VP16 is an interaction partner of dCas9-PIF. When crRNA and tracrRNA bind to dCas9, the protein is able to bind complementary DNA. When the system is exposed to red light (660 nm), the Phytochrome B receptor binds to the Phytochrome interaction actor (PIF). So this protein is being recruited by dCas9-PIF. The PhyB-PIF binding can be abolished by illumination with far-red light (740nm).
This system enables activation of gene expression induced by red light. For more information, have a look at our [http://2013.igem.org/Team:Freiburg/Project/induction#light light project page].
Data
Figure 1: SEAP levels for HEK293T-cells. The HEK293T-cells were transfected with dCas9-PIF6 and this part, PhyB-VP16. Medium was changed 5h post transfection. The cells were illuminated with red light (660nm, 20uE). Pre-illumination the cells were treated with 15uM phycocyanobilin in fresh media. Illumination started 24h post transfection and lasted for 48h, then a SEAP-assay (reporter) was performed. The bars on the left side show SEAP levels of the negative control that was transfected without the RNA plasmid. The bars on the right side show SEAP levels of constructs transfected with target plasmid. Red bars show cell-samples illuminated with activating red-light, white bars show samples illuminated with inactivating far-red light. Error bars show standard deviation of biological triplicates. An increasing SEAP-level after illumination can be observed if target plasmid is present.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 576
Illegal BglII site found at 1076
Illegal BamHI site found at 1158
Illegal XhoI site found at 1109
Illegal XhoI site found at 1128 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 1325
References
Müller, K. et al. (2013). A red/far-red light-responsive bi-stable toggle switch to control gene expression in mammalian cells. Nucleid Acid Research
Hirai, H. et al. (2010). Structure and functions of powerful transactivators: VP16, MyoD and FoxA. Int. J. Dev. Biol.
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