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Revision as of 12:58, 4 October 2013

uniCAS UV Light Switch Part I - Stimulator

CMV:HA-NLS-dCas9-Linker-UVR8-NLS:BGH
Function DNA binding 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]

Freiburg 2013 engineered a UVB light controlled mechanism to activate or repress gene expression. We fused this UVR8 receptor to dCas9. When crRNA and tracrRNA bind to Cas9, the protein is able to bind complementary DNA. If the system is exposed to UVB light (311 nm), the UVR8 receptor changes its configuration and recruits COP1 with its fused effector domain. Depending on which effector, gene expression can be activated or repressed on UVB light stimulus. Have a look at our [http://2013.igem.org/Team:Freiburg/Project/induction#light light project page].

Biology and Usage

Ultraviolet B (UVB) light can be perceived with photoreceptor UV resistance Locus 8 (UVR8) [1]. The receptor reacts best to light of the wavelenght 311 nm. UVR8 is fused to dCas9 with a short linker in between. Together with COP1, fused to an effector, it is possible to regulate gene expression by light induction.

In the absence of UVB light, UVR8 forms homodimers. This is its closed configuration. When UVB light is perceived, UVR8 monomerizes and changes into its open configuration. UVR8 is then able to recruit COP1 [2]. This interaction is disrupted when UVB radiation stops. COP1 can be fused to different effectors such as COP1-VP16, COP1-KRAB or COP1-G9a.

For this UVB light controled gene activation or repression you need a combination of these BioBricks: Cas9-UVR8, COP1-VP16 or COP1-KRAB / COP1-G9a and RNaimer.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 4815
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 576
    Illegal BglII site found at 900
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 4830

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

[2] Müller K., Engesser R., Schulz S., Steinberg T., Tomakidi P., Weber C. C., Ulm R., Timmer J., Zurbriggen M. D., Weber W. (2013). Multi-chromatic control of mammalian gene expression and signaling. Nucleic Acids Research Vol. 41, No. 12

[3] Favory J.-J., Stec A., Gruber H., Rizzini L., Oravecz A., Funk M., Albert A., Cloix C., Jenkins G. I., Oakeley E. J., Seidlitz H. K., Nagy F., Ulm R. (2009). Interaction of COP1 and UVR8 regulates UV-B-induced photomorphogenesis and stress acclimation in Arabidopsis. The EMBO Journal 28, 591-600