Difference between revisions of "Part:BBa K4586021"
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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>
 
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==
 
==Experimental Characterization==
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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.
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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 anti-CD19 and then measuring the specific concentration of the running part using Real-Time PCR as shown in the following figure.
 
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<br><br><br><br>
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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.
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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;
 
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"src="https://static.igem.wiki/teams/4586/wiki/parts-experiments/digestion-2.png">
 
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<br><br>
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After the ligation step, we did culture of the ligated product to specifically select the optimum colonies to screen it using Colony PCR to make sure that our parts were correctly ligated in the plasmid.
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The cell culture plate of transformed pCDNA vector containing insert parts is shown in the following figure.
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This plasmid contains
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1-Syn-notch (CD8 alpha-his tag-Anti CD19-mouse notch core-ZF21.16\VP64))
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2-Booster gene 1 (SDC4, STEAP3)
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3-Booster gene 2 (NAdB)
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"src="https://static.igem.wiki/teams/4586/wiki/parts/vector-1.png">
 
<p class=MsoNormal align=center style='text-align:left;border:none;width:98% ;justify-content:center;'><span
 
<p class=MsoNormal align=center style='text-align:left;border:none;width:98% ;justify-content:center;'><span
 
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Latest revision as of 14:02, 12 October 2023


VP64

Part Description

This parts codes for VP64 which is transcriptional activator peptide formed of two components, first four tandem repeats of VP16 (Herpes simplex viral protein) second, amino acids 437-447*. This transcription module is a classic molecular biology tool due to its strong transcriptional activity that can be used for conditional gene expression by fusing to it another protein domain as ZF peptide that can bind near the promoter of the gene of interest.

Usage

We implemented this part in our design to trigger the transcriptional activity of ZF21.61 minCMV promoter that control the expression of our therapeutic agent or cargo (Cas12k). Therefore, VP64 share in forming the composite part ZF21.16-VP64 that represent the internal domain of our syn notch receptor which design to be activated after cell to cell interaction between MSC and B-cell receptor as shown in figure 1.

Figure 1: This figure illustrates the design of our biological circuit expressing our therapeutic agent under the control of the VP64 transcription module that regulates the activity of the ZF21.16minCMV promoter.


Literature Characterization

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

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

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 anti-CD19 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)



After the ligation step, we did culture of the ligated product to specifically select the optimum colonies to screen it using Colony PCR to make sure that our parts were correctly ligated in the plasmid. The cell culture plate of transformed pCDNA vector containing insert parts is shown in the following figure. This plasmid contains 1-Syn-notch (CD8 alpha-his tag-Anti CD19-mouse notch core-ZF21.16\VP64)) 2-Booster gene 1 (SDC4, STEAP3) 3-Booster gene 2 (NAdB)

References

Alerasool, N., Leng, H., Lin, Z. Y., Gingras, A. C., & Taipale, M. (2022). Identification and functional characterization of transcriptional activators in human cells. Molecular cell, 82(3), 677-695. 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]