Difference between revisions of "Part:BBa K4839011"

 
 
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In order to prove our concept, we construct an expression cassette of GAL4 BS-UAS-GFP-pGK-mcherry. The GAL4 BS (GAL4 binding site) can bind with the GAL4 element and the UAS element can bind with the VP64 element. Once the SNIPR receptor recognize and target to the GPC3 receptor of Huh7 or THP-1 cancer cell, it will mediate downstream signal transduction and cut off the GAL4-VP64 factor. The GAL4-VP64 will bind to the GAL4 binding site and trigger the binding of VP64 and UAS element. The UAS promoter then activated by this transcriptional factor and start the transcription.  
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In order to prove our concept, we construct an expression cassette of GAL4 BS-UAS-GFP-pGK-mcherry. The GAL4 BS (GAL4 binding site) can bind with the GAL4 element and the UAS element can bind with the VP64 element. Once the SNIPR receptor recognize and target to the GPC3 receptor of Huh7 or THP-1 cancer cell, it will mediate downstream signal transduction and cut off the GAL4-VP64 factor. The GAL4-VP64 will bind to the GAL4 binding site and trigger the binding of VP64 and UAS element. The UAS promoter then activated by this transcriptional factor and start the transcription. (Figure1)
  
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===Usage and Biology===
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<html><img src="https://static.igem.wiki/teams/4839/wiki/parts-files/9-1.png" width="650"</html>
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<p align="center">Figure1. The prove-of-concept of our SNIPR design</p>
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<p>[1] Zhu I , Liu R , Wittsten A H ,et al.Design and modular assembly of synthetic intramembrane proteolysis receptors for custom gene regulation in therapeutic cells[J]. 2021.DOI:10.1101/2021.05.21.445218.</p>
  
 
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<partinfo>BBa_K4839011 parameters</partinfo>
 
<partinfo>BBa_K4839011 parameters</partinfo>
 
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===Contribution===
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Team: Duke iGEM 2024
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Our team used the Gal4 UAS sequence to verify the activation of the synNotch platform. Our Gal4 UAS reporter system contained BFP in place of GFP and reported higher median BFP fluorescence when co-transfected with constitutively expressed Gal4-VP64 (<b>Figure 1</b>).
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<div style="text-align: center;">
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<img src="https://static.igem.wiki/teams/5198/registry/bba-k3132000-fig1.webp" alt="Description of the image" style="width: 400px; height: auto;">
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<p><b>Figure 1.</b> Representative histogram of median BFP fluorescence in HEK293T cells co-expressing Gal4-VP64 activator and Gal4 UAS-BFP reporter system. The negative control (NC) cells were transfected with only the Gal4 UAS-BFP reporter system and show lower median fluorescence. </p>
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The results demonstrate the ability of this sequence to respond to Gal4-VP64 even when expressed independently of synNotch. For this reason, this part can be confidently used to drive inducible expression of custom gene programs.
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Latest revision as of 11:43, 2 October 2024


UAS (VP64 binding element)


In order to prove our concept, we construct an expression cassette of GAL4 BS-UAS-GFP-pGK-mcherry. The GAL4 BS (GAL4 binding site) can bind with the GAL4 element and the UAS element can bind with the VP64 element. Once the SNIPR receptor recognize and target to the GPC3 receptor of Huh7 or THP-1 cancer cell, it will mediate downstream signal transduction and cut off the GAL4-VP64 factor. The GAL4-VP64 will bind to the GAL4 binding site and trigger the binding of VP64 and UAS element. The UAS promoter then activated by this transcriptional factor and start the transcription. (Figure1)


Figure1. The prove-of-concept of our SNIPR design

[1] Zhu I , Liu R , Wittsten A H ,et al.Design and modular assembly of synthetic intramembrane proteolysis receptors for custom gene regulation in therapeutic cells[J]. 2021.DOI:10.1101/2021.05.21.445218.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Contribution

Team: Duke iGEM 2024
Our team used the Gal4 UAS sequence to verify the activation of the synNotch platform. Our Gal4 UAS reporter system contained BFP in place of GFP and reported higher median BFP fluorescence when co-transfected with constitutively expressed Gal4-VP64 (Figure 1).

Description of the image

Figure 1. Representative histogram of median BFP fluorescence in HEK293T cells co-expressing Gal4-VP64 activator and Gal4 UAS-BFP reporter system. The negative control (NC) cells were transfected with only the Gal4 UAS-BFP reporter system and show lower median fluorescence.

The results demonstrate the ability of this sequence to respond to Gal4-VP64 even when expressed independently of synNotch. For this reason, this part can be confidently used to drive inducible expression of custom gene programs.