Part:BBa_K3242006
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.
Within the GAL4 regulatory protein, the GAL4 binding domain (GAL4BD) is located at the N-terminus of the polypeptide. The GAL4 binding domain binds to a DNA sequence, known as a galactose upstream activating sequence (UAS) [2].
VP16 is known to be a transcription factor derived from the herpes simplex virus. 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) [3].
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
The 2019 UGA iGEM team cotransformed this part with a 1xUAS-BS3 resistance reporter gene into agrobacterium. Upon a successful transformation, the team infiltrated Nicotiana benthamiana and observed the expression of the BS3 resistance gene.
In the figures that follow, the results of the transfection of GAL4BD-VP16 and 1XUAS-BS3 into Nicotiana benthamiana can be seen. When doing our infiltrations, we had 5 different spots on the leaf. Here is the Key:
Infiltration Spots - Key
- BS3 in pGWB2
- Yellow Fluorescent Protein (YFP)
- Gal4BD-VP16
- BS3 in pGWB1
- BS3 in pGWB1 + Gal4BD-VP16
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Based on these results, it is clear that the control part works as expected. Ideally, the only spot where any sort of cell death should be present is spot 5. However, realistically, some cell death should be apparent in the spots that contain only BS3, either in pGWB1 (spot 4) or pGWB2 (spot 1). This is due to any “leaky” expression that might be occurring in the plant. In all three trials, the greatest amount of cell death was observed in spot 5, followed by spot 4, and then lastly spot 1. This is consistent with what we expected. We expected the greatest amount of cell death in spot 5 because the Gal4BD-VP16 transcriptional factor should be driving strong downstream expression of BS3. Background BS3 expression should be higher in pGWB2 than in pGWB1 due to the presence of the constitutive p35S promoter. This is evident through spot 4 showing higher levels of cell death than spot 1. Finally, we know that the control part functions properly because there is no cell death in spot 3 (where Gal4BD-VP16 is infiltrated on its own), but there is cell death in spot 5 (where Gal4BD-VP16 is infiltrated in conjunction with 1xUAS-BS3).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 218
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 343
Illegal SapI site found at 198
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