Difference between revisions of "Part:BBa K1150026"
 
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! colspan="2" style="background:#FFBF00;"|uniCAS red Light Switch Part II - Activator
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! colspan="2" style="background:#FFBF00;"|CMV:PhyB-Linker-VP16-NLS:BGH
 
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|'''Function'''
 
|'''Function'''
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|[http://2013.igem.org/Team:Freiburg Freiburg 2013]
 
|[http://2013.igem.org/Team:Freiburg Freiburg 2013]
 
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==Usage and Biology==
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Fusion protein of [https://parts.igem.org/Part:BBa_K1150004 PhyB] and [https://parts.igem.org/Part:BBa_K1150001 VP16].
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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 [https://parts.igem.org/Part:BBa_K1150000 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 [https://parts.igem.org/Part:BBa_K1150025 dCas9-PIF]. The PhyB-PIF binding can be abolished by illumination with far-red light (740nm).
  
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===Usage and Biology===
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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].
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Fusion protein of [https://parts.igem.org/Part:BBa_K1150004|PhyB] and [https://parts.igem.org/Part:BBa_K1150001|VP16].
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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.
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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.
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===References===
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==Data==
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Müller, K.; Engesser, R.; Metzger, S.; Schulz, S.; Kampf, M. M.; Busacker, M. et al. (Nucleid Acid Research, 2013): A red/far-red light-responsive bi-stable toggle switch to control gene expression in mammalian cells.
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[[File:Freiburg2013_light_results_red2.png|left|300px]]<br>
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'''Figure 1:''' SEAP levels for HEK293T-cells.  
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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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<span class='h3bb'>Sequence and Features</span>
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<span class='h3bb'>
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==Sequence and Features==
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</span>
 
<partinfo>BBa_K1150026 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1150026 SequenceAndFeatures</partinfo>
  
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==References==
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<small>
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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 <br>
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Hirai, H. et al. (2010). Structure and functions of powerful transactivators: VP16, MyoD and FoxA. Int. J. Dev. Biol.
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</small>
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 01:54, 5 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 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

Freiburg2013 light results red2.png

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.