Difference between revisions of "Part:BBa K1150030"

Revision as of 21:18, 4 October 2013

uniCAS UV Light Switch Part II - Activator

CMV:COP1-VP16:BGH
Function	Activation domain of UVB light induced gene expression control
Use in	Mammalians
RFC standard	RFC 25
Backbone	pSB1C3
Submitted by	[http://2013.igem.org/Team:Freiburg Freiburg 2013]

The fusion protein COP1-VP16 is an interaction partner of dCas9-UVR8. It's a part of the UVB light controlled mechanism to activate gene expression. First the dCas9-UVR8 fusion protein binds specifically to a DNA target sequence. If the system is exposed to UVB light (311 nm), the UVR8 receptor of UV changes its configuration and recruits this COP1-VP16 protein. This activates gene expression induced by UVB light.

For more information have a look at our [http://2013.igem.org/Team:Freiburg/Project/induction#light light project page].

Biology and Usage

E3-ubiquitin ligase Constitutively Photomorphogenic 1 (COP1) is the interaction partner of ultraviolet B (UVB) light receptor UVR8 [1]. When UVB light is perceived by the UVR8 photoreceptor it monomerizes and therefore changes in its open confuguration. Not till then it recruits COP1. For this UVB light controled gene activation system you need a combination of these BioBricks: dCas9-UVR8, COP1-VP16 and RNAimer.

Data

Figure 1:SEAP levels for HEK293T-cells. The HEK293T-cells were transfected with plasmids containig dCas9-UVR8, this plasmid containing COP1-VP16, the RNAimer target plasmid and a SEAP-reporter plasmid. The bars on the left side show SEAP levels of the negative control that was transfected without the targeting plasmid (RNAimer). Medium was changed 5h post PEI transfection. Cells were illuminated with UVB light (311nm, 5uE) for 24h. After illumination a SEAP-assay was performed. The bars on the right side show SEAP levels of constructs transfected with target plasmid containing 3 different target sites. Violet bars show cell-samples illuminated with activating UVB light, white bars show samples that were kept in darkness. Error bars show standard deviation of biological triplicates. A two-fold increase of SEAP-level after illumination can be observed, if the 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
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 1373
Illegal BsaI.rc site found at 1450
Illegal SapI.rc site found at 962

References

[1] Rizzini L., Favory J.-J., Cloix C., Faggionato D., O'Hara A., Kaiserli E., Baumeister R., Schäfer E., Nagy F., Jenkins G. I., Ulm R. (2011). Perception of UV-B by the Arabidopsis UVR8 protein. Science 332

@@ Line 22: / Line 22: @@
 |}
-The fusion protein COP1-VP16 is an interaction partner of [https://parts.igem.org/Part:BBa_K1150029 dCas9-UVR8]. It's a part of the UVB light controlled mechanism to activate gene expression. First the dCas9-UVR8 fusion protein binds specifically to a DNA target sequence. If the system is exposed to UVB light (311 nm), the UVR8 receptor of UV changes its configuration and recruits this COP1-VP16 protein. This activates gene expression induced by UVB light. Have a look at our [http://2013.igem.org/Team:Freiburg/Project/induction#light light project page].
+The fusion protein COP1-VP16 is an interaction partner of [https://parts.igem.org/Part:BBa_K1150029 dCas9-UVR8]. It's a part of the UVB light controlled mechanism to activate gene expression. First the dCas9-UVR8 fusion protein binds specifically to a DNA target sequence. If the system is exposed to UVB light (311 nm), the UVR8 receptor of UV changes its configuration and recruits this COP1-VP16 protein. This activates gene expression induced by UVB light.
+For more information have a look at our [http://2013.igem.org/Team:Freiburg/Project/induction#light light project page].
@@ Line 34: / Line 36: @@
 [[File:Freiburg2013_light_results_uv2.png|left|300px]]<br>
 '''Figure 1:'''SEAP levels for HEK293T-cells.
-The HEK293T-cells were transfected with plasmids containig dCas9-UVR8, this plasmid containing COP1-VP16, the RNAimer target plasmid and a SEAP-reporter plasmid. The bars on the left side show SEAP levels of the negative control that was transfected without the targeting plasmid (RNAimer). Medium was changed 5h post PEI transfection. Cells were illuminated with UV-b light (311nm, 5uE) for 24h. After illumination a SEAP-assay was performed. The bars on the right side show SEAP levels of constructs transfected with target plasmid containing 3 different target sites. Violet bars show cell-samples illuminated with activating UV-b light, white bars show samples that were kept in darkness. Error bars show standard deviation of biological triplicates. An increasing SEAP-level after illumination can be observed, if target plasmid is present. A two-fold induction can be observed.
+The HEK293T-cells were transfected with plasmids containig dCas9-UVR8, this plasmid containing COP1-VP16, the RNAimer target plasmid and a SEAP-reporter plasmid. The bars on the left side show SEAP levels of the negative control that was transfected without the targeting plasmid (RNAimer). Medium was changed 5h post PEI transfection. Cells were illuminated with UVB light (311nm, 5uE) for 24h. After illumination a SEAP-assay was performed. The bars on the right side show SEAP levels of constructs transfected with target plasmid containing 3 different target sites. Violet bars show cell-samples illuminated with activating UVB light, white bars show samples that were kept in darkness. Error bars show standard deviation of biological triplicates. A two-fold increase of SEAP-level after illumination can be observed, if the target plasmid is present.
 <span class='h3bb'>
 ==Sequence and Features==