Difference between revisions of "Part:BBa J176013"

(→‎Usage and Biology)
 
(15 intermediate revisions by 3 users not shown)
Line 6: Line 6:
 
===Usage and Biology===
 
===Usage and Biology===
  
−
* Chassis: mammalian cells
+
[[Image:KAH_VP64.png|thumb|left|350px|'''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
 
* Mammalian expression vector required
 
* Protein domain; requires promoter, start codon, stop codon, and polyA signal for proper expression
 
* 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<br>
+
* 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<br>  
  
  
−
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 linkers. When fused to another protein domain that can bind near the promoter of a gene, VP64 acts as a strong transcriptional activator.
+
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 [https://parts.igem.org/Part:BBa_J176013:Design 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.
 +
<html><div align="center"style="border:solid #17252A; width:50%;float:center;"><img style="                              max-width:850px;
 +
width:75%;
 +
height:auto;
 +
position: relative;
 +
top: 50%;
 +
left: 35%;
 +
transform: translate( -50%);
 +
padding-bottom:25px;
 +
padding-top:25px;
 +
"src="https://static.igem.wiki/teams/4586/wiki/literature-characterisation-parts/vp64.png">
 +
<p class=MsoNormal align=center style='text-align:left;border:none;width:98% ;justify-content:center;'><span
 +
lang=EN style='font-size:11.0pt;line-height:115%'>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. </span></p></div></html>
 +
==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).
 +
<html><div align="center"style="border:solid #17252A; width:100%;float:center;"><img style="                              max-width:850px;
 +
width:90%;
 +
height:auto;
 +
position: relative;
 +
top: 50%;
 +
left: 45%;
 +
transform: translate( -50%);
 +
padding-bottom:25px;
 +
padding-top:25px;
 +
"src="https://static.igem.wiki/teams/4586/wiki/modeling/21.png">
 +
<p class=MsoNormal align=center style='text-align:left;border:none;width:98% ;justify-content:center;'><span
 +
lang=EN style='font-size:11.0pt;line-height:115%'>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).  </span></p></div></html>
 +
==Experimental Characterization by AFCM-Egypt==
 +
In order to amplify this DNA part, we used PCR amplification to reach the desired concentration to complete our experiments using specific forward and reverse primers, running the parts on gel electrophoresis as this part presents in lane (P1) including CD8 alpha-his tag-mouse notch core-ZF21.16\VP64, and then measuring the specific concentration of the running part using Real-Time PCR as shown in the following figure.
 +
<html><div align="center"style="border:solid #17252A; width:80%;float:center;"><img style="                              max-width:850px;
 +
width:100%;
 +
height:auto;
 +
position: relative;
 +
top: 50%;
 +
left: 50%;
 +
transform: translate( -50%);
 +
padding-bottom:25px;
 +
padding-top:25px;
 +
"src="https://static.igem.wiki/teams/4586/wiki/parts-experiments/pcr-ampli.png">
 +
<p class=MsoNormal align=center style='text-align:left;border:none;width:98% ;justify-content:center;'><span
 +
lang=EN style='font-size:11.0pt;line-height:115%'>
  
−
===Characterization===
+
</span></p></div></html>
−
<b>VP64 activity is sensitive to promoter proximity.</b><br>
+
<br><br><br><br>
−
In our hands, VP64 activity is maximized when it is targeted to DNA sequences that are ~40-80 base pairs away from a minimal promoter. When fused to the Gal4 DNA binding domain (<partinfo>BBa_J176020</partinfo>) and targeted it to Gal (UAS) DNA elements (<partinfo>BBa_J176019</partinfo>) that are assembled immediately adjacent to the HSVtkTATA minimal promoter (<partinfo>BBa_J176011</partinfo>), we observe weak activation, and perhaps repression, of a fluorescent reporter gene. In contrast, when a small spacer (<partinfo>BBa_J176039</partinfo>) is inserted between the Gal4 binding sites and the promoter, reporter gene activity greatly increases.
+
We performed the double digestion method for this part in the prefix and suffix with its specific restriction enzyme and applied this part to gel electrophoresis as shown in the following figure in lane (P1)
 +
<html><div align="center"style="border:solid #17252A; width:80%;float:center;"><img style="                              max-width:850px;
 +
width:100%;
 +
height:auto;
 +
position: relative;
 +
top: 50%;
 +
left: 50%;
 +
transform: translate( -50%);
 +
padding-bottom:25px;
 +
padding-top:25px;
 +
"src="https://static.igem.wiki/teams/4586/wiki/parts-experiments/digestion-2.png">
 +
<p class=MsoNormal align=center style='text-align:left;border:none;width:98% ;justify-content:center;'><span
 +
lang=EN style='font-size:11.0pt;line-height:115%'>
 +
 
 +
</span></p></div></html>
  
−
(Data will be posted soon)
 
  
 
<!-- -->
 
<!-- -->
Line 29: Line 86:
 
<partinfo>BBa_J176013 parameters</partinfo>
 
<partinfo>BBa_J176013 parameters</partinfo>
 
<!-- -->
 
<!-- -->
 +
 +
==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.
 +
 +
[[Image:T--SMMU-China--TFs Fluorescence.png|middle|800px|'''Figure. 1 mCherry expression activated by the transcription factors''']]
 +
<B>Figure. 1</B> mCherry expression activated by the transcription factors
 +
 +
[[Image:T--SMMU-China--TFs Data.png|middle|600px|'''Figure. 2 The statistical result of all the TFs-Promoters pairs'']]
 +
<B>Figure. 2</B> The statistical result of all the TFs-Promoters pairs

Latest revision as of 20:53, 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).

Experimental Characterization by AFCM-Egypt

In order to amplify this DNA part, we used PCR amplification to reach the desired concentration to complete our experiments using specific forward and reverse primers, running the parts on gel electrophoresis as this part presents in lane (P1) including CD8 alpha-his tag-mouse notch core-ZF21.16\VP64, and then measuring the specific concentration of the running part using Real-Time PCR as shown in the following figure.





We performed the double digestion method for this part in the prefix and suffix with its specific restriction enzyme and applied this part to gel electrophoresis as shown in the following figure in lane (P1)


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