Composite

Part:BBa_K3242006

Designed by: Lui Suzuki-Williams   Group: iGEM19_UGA   (2019-10-19)
Revision as of 00:35, 21 October 2019 by LuiSuzukiWilliams (Talk | contribs) (→‎Usage and Biology)


GAL4BD - VP16

GAL4BD - VP16 is a modified version of the Gal4 transcription factor found in yeast [1]. With this hybrid transcription factor, the UGA iGEM team developed a UAS-GAL4 expression system. GAL4BD-VP16 would allow for the constitutive expression of a reporter gene that follows an upstream activating sequence.

Usage and Biology

GAL4BD - VP16 is merely a conjunction of the GAL4 binding domain and the VP16 transcriptional activator (derived from Herpes simplex virus). This part is a strong transcriptional factor that binds to an upstream activating sequence (UAS) that generally precedes some downstream gene of interest. It can be used in inducible synthetic constructs, wherein GAL4BD-VP16 activates the expression of some downstream gene of interest.

Figure 1. Model of the GAL4 transcription factor and the transcriptional activation of a gene. The GAL4 Binding Domain recognizes and binds to an upstream activating DNA sequence.

Within the GAL4 regulatory protein, the GAL4 binding domain (GAL4BD) is located at the N-terminus of the polypeptide [2]. The GAL4 binding domain binds to a DNA sequence, known as a galactose upstream activating sequence (UAS).

Figure 2. Model of the VP16 transcription factor. The VP16 pictured is recruiting other transcriptional factors for the initiation of transcription.

VP16 is known to be a transcription factor derived from the herpes simplex virus [3]. Within VP16 is the transactivating domain (TAD), and it is regarded as an extremely potent TAD. The transactivating domain of VP16 interacts with various other transcriptional machinery (e.g. basal transcription factors) to promote transcription of immediate-early genes. VP16 TAD plays a crucial for the assembly of the pre-initiation complex, and it is versatile in its ability to be fused to a wide range of DNA binding domains (e.g. GAL4BD).

Figure 3. Model of the GAL4/UAS System. The model displays the interaction between the GAL4 transcription factor and a UAS DNA sequence that precedes a gene of interest.

Together, GAL4BD and VP16 TAD provide strong gene expression in a GAL4-UAS modified system. The GAL4-UAS system was developed in Drosophila for the selective expression of a researcher’s gene of interest. Figure 3 is a model of the system in Drosophila, but UGA iGEM is applying this system for use in bacteria and plants.







Results



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 218
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 343
    Illegal SapI site found at 198


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Categories
Parameters
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