Difference between revisions of "Part:BBa J176013"

Revision as of 19:53, 11 October 2023

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.

Literature Characterization by AFCM-Egypt

vp64 were transfected into HEK293T reporter cells and treated with abscisic acid or DMSO for 48 h.

Statistical significance was calculated with one-way ANOVA, using Dunnett’s multiple testing correction which showed that GFP expression was higher with VP64 than with any other promoter.

charactrization by mathematical modeling by AFCM-Egypt

This internal domain is activated after cell to cell interaction between MSC and B-cell receptor. That leads to increase of transcription factor VP64 that triggers the expression of the internal circuit that secretes the exosome's cargo then cargo loading to exosomes through the loading system (CD63-L7Ae).

As the expression of the internal domain increases (represented as red line), the transcription factor VP64 increases, increasing the expression of the internal circuit for the exosome's cargo to finally produce modified exosomes (represented in blue line).

Sequence and Features

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