Difference between revisions of "Part:BBa J176013"
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<partinfo>BBa_J176013 parameters</partinfo>
 
<partinfo>BBa_J176013 parameters</partinfo>
 
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==Characterization: SMMU-China 2019==
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VP64 is a strong transcriptional activator, however it can only act when fused to a DNA binding domain. Here, we fused this part to four different DNA binding domains separately and measured their activating effect. The four transcription factors (TFs) that we constructed are Gal4-VP64 (BBa_K3132000), PIP-VP64 (BBa_K3132003), ZF21-16-VP64 (BBa_K3132002), and ZF43-8-VP64 (BBa_K3132001). The TFs and their corresponding promoters (see in part pages of the TFs) were co-transfected in HEK293T cells. Cells that were transfected only with promoter-mCherry plasmid were used as a negative control. The photos taken by fluorescence microscopy and the statistical results are shown in figure. 1 and figure. 2. This panel of TFs showed different activities, and provide more choices for users to choose from.
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[[Image:T--SMMU-China--TFs Fluorescence.png|middle|800px|'''Figure. 1 mCherry expression activated by the transcription factors''']]
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<B>Figure. 1</B> mCherry expression activated by the transcription factors
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[[Image:T--SMMU-China--TFs Data.png|middle|600px|'''Figure. 2 The statistical result of all the TFs-Promoters pairs'']]
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<B>Figure. 2</B> The statistical result of all the TFs-Promoters pairs

Revision as of 12:01, 17 October 2019

VP64

Tetrameric VP16 transcription activator domain

Usage and Biology

The VP64 Activation Domain Module is often fused to DNA binding domains to make strong synthetic transcription factors. Figure adapted from Beerli et al., 1998.
  • Mammalian expression vector required
  • Protein domain; requires promoter, start codon, stop codon, and polyA signal for proper expression
  • VP64 doesn't do much as a free-floating domain. Fuse it to a DNA binding domain for the best results
  • Uniprot entry UL48 (VP16) - VP64 is a 4x tandem array of amino acids 438-448


VP64 is a transcriptional activator composed of four tandem copies of VP16 (Herpes Simplex Viral Protein 16, amino acids 437-447*: DALDDFDLDML) connected with glycine-serine (GS) linkers. When fused to another protein domain that can bind near the promoter of a gene, VP64 acts as a strong transcriptional activator. This module is a classic molecular biology tool. See the References section for some history on VP16 and VP64. *Note: numbering excludes the starting methionine so the first a.a. is actually 438, consistent with the Uniprot entry UL48.


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]


Characterization: SMMU-China 2019

VP64 is a strong transcriptional activator, however it can only act when fused to a DNA binding domain. Here, we fused this part to four different DNA binding domains separately and measured their activating effect. The four transcription factors (TFs) that we constructed are Gal4-VP64 (BBa_K3132000), PIP-VP64 (BBa_K3132003), ZF21-16-VP64 (BBa_K3132002), and ZF43-8-VP64 (BBa_K3132001). The TFs and their corresponding promoters (see in part pages of the TFs) were co-transfected in HEK293T cells. Cells that were transfected only with promoter-mCherry plasmid were used as a negative control. The photos taken by fluorescence microscopy and the statistical results are shown in figure. 1 and figure. 2. This panel of TFs showed different activities, and provide more choices for users to choose from.

Figure. 1 mCherry expression activated by the transcription factors 

Figure. 1 mCherry expression activated by the transcription factors

'Figure. 2 The statistical result of all the TFs-Promoters pairs 

Figure. 2 The statistical result of all the TFs-Promoters pairs