Difference between revisions of "Part:BBa K1150026"
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==Data== | ==Data== | ||
[[File:Freiburg2013_light_results_red2.png|left|300px]]<br> | [[File:Freiburg2013_light_results_red2.png|left|300px]]<br> | ||
| − | '''Figure 1:''' SEAP levels for HEK293T-cells. | + | '''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 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 targeting plasmid | + | The bars on the left side show SEAP levels of the negative control that was transfected without the targeting plasmid https://parts.igem.org/Part:BBa_K1150034. 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. |
<span class='h3bb'> | <span class='h3bb'> | ||
Revision as of 15:57, 4 October 2013
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 Cas9, 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 it recruits the dCas9-PIF protein. 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. 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 targeting plasmid https://parts.igem.org/Part:BBa_K1150034. 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.