Difference between revisions of "Part:BBa K1150026"

Revision as of 20:26, 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 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

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE 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
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE 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.

@@ Line 24: / Line 24: @@
 ==Usage and Biology==
 Fusion protein of [https://parts.igem.org/Part:BBa_K1150004 PhyB] and [https://parts.igem.org/Part:BBa_K1150001 VP16].
-This fusion protein PhyB-VP16 is an interaction partner of [https://parts.igem.org/Part:BBa_K1150025 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 [https://parts.igem.org/Part:BBa_K1150025 dCas9-PIF] protein.
+This fusion protein PhyB-VP16 is an interaction partner of [https://parts.igem.org/Part:BBa_K1150025 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 this protein is being recruited by [https://parts.igem.org/Part:BBa_K1150025 dCas9-PIF]. The PhyB-PIF binding can be abolished by illumination with far-red light (740nm).
-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].
+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].
@@ Line 31: / Line 32: @@
 [[File:Freiburg2013_light_results_red2.png|left|300px]]<br>
 '''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.
+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 [https://parts.igem.org/Part:BBa_K1150034 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.
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