Difference between revisions of "Part:BBa K3242006"

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===Usage and Biology===
 
===Usage and Biology===
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[[File:GAL4BD.png|300px|thumb|right|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 [4].]]
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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.
 
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.
  
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[[File:GAL4BD.png|300px|thumb|left|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.]]
 
  
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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).
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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].
 +
 
 +
[[File:VP16.jpeg|300px|thumb|left|Figure 2. Model of the VP16 transcription factor. The VP16 pictured is recruiting other transcriptional factors for the initiation of transcription [3].]]
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[[File:GAL4 UAS.jpeg|300px|thumb|right|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 [1].]]
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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].
  
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[[File:VP16.jpeg|300px|thumb|right|Figure 2. Model of the VP16 transcription factor. The VP16 pictured is recruiting other transcriptional factors for the initiation of transcription.]]
 
  
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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).
 
  
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[[File:GAL4 UAS.jpeg|300px|thumb|left|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.
 
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.
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===Results===
 
===Results===
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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.
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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:
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<strong>Infiltration Spots - <i>Key</i></strong>
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<ol>
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<li>BS3 in pGWB2 (contains a constitutive P35s Promoter)</li>
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<li>Yellow Fluorescent Protein (YFP)</li>
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<li>Gal4BD-VP16</li>
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<li>BS3 in pGWB1 (does not contain a constitutive P35s Promoter)</li>
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<li>BS3 in pGWB1 + Gal4BD-VP16</li>
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</ol>
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https://2019.igem.org/wiki/images/f/f6/T--UGA--Trials.png
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<p>
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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, some cell death was observed in the experimental spots that contain only BS3, either in pGWB1 (spot 4) or pGWB2 (spot 1). This was likely due to “leaky” expression occurring in the plant. In all three trials, the greatest amount of cell death was observed in spot 5, followed by spot 1, and then lastly spot 4. This was consistent with our expectations. We expected the greatest amount of cell death in spot 5 because the Gal4BD-VP16 transcriptional factor should be driving a strong downstream expression of BS3. Background BS3 expression was also expected to be higher in pGWB2 than in pGWB1 due to the presence of a constitutive p35S promoter. This is evident as spot 1 displayed higher levels of cell death than spot 4. Finally, we confirmed that the control part functions properly because there was no observed cell death in spot 3 (where Gal4BD-VP16 is infiltrated on its own), but there was observed cell death in spot 5 (where Gal4BD-VP16 is infiltrated in conjunction with 1xUAS-BS3).
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<br>
 
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As discussed above, the 2019 UGA iGEM team was able to validate the function of the GAL4BD-VP16 part. Additionally, our sequencing was confirmed through Europhins when the part was inserted into a gateway binary vector (pGWB2). Due to the availability of our resources, the UGA iGEM team was only able to validate this part through its ability to drive expression of a reporter gene (BS3). In the future, we intend to test our GAL4BD-VP16 transcription factor on other UAS-reporter genes, in an effort to further validate its function as a versatile and strong transcription factor. The UGA iGEM team is also developing a 6xUAS-BS3 gene to generate a stronger expression of cell death with the GAL4BD-VP16 part.
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<br><span class='h3bb'>Sequence and Features</span>
 
<br><span class='h3bb'>Sequence and Features</span>

Latest revision as of 01:38, 22 October 2019


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

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 [4].

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].

Figure 2. Model of the VP16 transcription factor. The VP16 pictured is recruiting other transcriptional factors for the initiation of transcription [3].
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 [1].


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

  1. BS3 in pGWB2 (contains a constitutive P35s Promoter)
  2. Yellow Fluorescent Protein (YFP)
  3. Gal4BD-VP16
  4. BS3 in pGWB1 (does not contain a constitutive P35s Promoter)
  5. BS3 in pGWB1 + Gal4BD-VP16

T--UGA--Trials.png

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, some cell death was observed in the experimental spots that contain only BS3, either in pGWB1 (spot 4) or pGWB2 (spot 1). This was likely due to “leaky” expression occurring in the plant. In all three trials, the greatest amount of cell death was observed in spot 5, followed by spot 1, and then lastly spot 4. This was consistent with our expectations. We expected the greatest amount of cell death in spot 5 because the Gal4BD-VP16 transcriptional factor should be driving a strong downstream expression of BS3. Background BS3 expression was also expected to be higher in pGWB2 than in pGWB1 due to the presence of a constitutive p35S promoter. This is evident as spot 1 displayed higher levels of cell death than spot 4. Finally, we confirmed that the control part functions properly because there was no observed cell death in spot 3 (where Gal4BD-VP16 is infiltrated on its own), but there was observed cell death in spot 5 (where Gal4BD-VP16 is infiltrated in conjunction with 1xUAS-BS3).

As discussed above, the 2019 UGA iGEM team was able to validate the function of the GAL4BD-VP16 part. Additionally, our sequencing was confirmed through Europhins when the part was inserted into a gateway binary vector (pGWB2). Due to the availability of our resources, the UGA iGEM team was only able to validate this part through its ability to drive expression of a reporter gene (BS3). In the future, we intend to test our GAL4BD-VP16 transcription factor on other UAS-reporter genes, in an effort to further validate its function as a versatile and strong transcription factor. The UGA iGEM team is also developing a 6xUAS-BS3 gene to generate a stronger expression of cell death with the GAL4BD-VP16 part.

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