Difference between revisions of "Part:BBa K4586002"

(→‎Part Description)
(→‎Experimental Characterization)
 
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==Usage==
 
==Usage==
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This part is implemented in our design to represent the internal domain of the syn notch receptor, which controls the level of our therapeutic agent by releasing the transcription factor VP64, which triggers the expression of the internal circuit that secretes the exosome's cargo, which is regulated by ZF 21-16 promoter activity, which is composed of three main components.
+
We used this part in our design to form the internal domain of our Syn-Notch receptor (BBa_K4586022).
 +
This internal domain is released after receptor activation and guided by the ZF21.16 to the target promoter that regulates the levels of expression of our therapeutic agent as shown in figure 1.
 +
<html><div align="center"style="border:solid #17252A; width:100%;float:center;"><img style="                              max-width:850px;
 +
width:100%;
 +
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/parts/3-21-25.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%'>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.   </span></p></div></html>
  
 +
==Literature Characterization==
 +
The study used ZF21 antibody for detection of ZF21 by Western blot at 1 µg/mL. For immunofluorescence, start at 20 µg/mL.
 +
<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/zf21-16.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%'> (A)western blot analysis of ZF21 in 3T3 cell tissue lysate with ZF 21 antibodies in abscence of blocking peptide while (B) the same western blot analysis in presence of blocking peptide </span></p></div></html>
 +
 +
==charactrization of mathematical modeling==
 +
We are using mathematical modeling to simulate the effect of activation on the internal domain on the level of expression of our engineered exosomes so on activation of Zinc finger proteins (ZF21.16) which represent a part of the internal domain that leads to stimulation of the whole internal domain.
 +
<html><div align="center"style="border:solid #17252A; width:100%;float:center;"><img style="                              max-width:850px;
 +
width:80%;
 +
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/modeling/zd21-16-1.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%'> The graph shows the relation between activation of the internal domain of synthetic notch and increased level of engineered exosomes.</span></p></div></html>
 +
<br><br>
 +
comparison between the types of internal domains of different receptors  based on their ability for production of exosomes.
 +
<br>
 +
1)We modeled the kinetics of chimeric antigen receptor (CAR) to explain production of engineered exosomes from MSC, but it’s concluded that it wasn’t efficient as there’s nearly detected signal of exosomes production as its internal domain couldn’t be modified to be used in MSC
 +
<html><div align="center"style="border:solid #17252A; width:100%;float:center;"><img style="                              max-width:850px;
 +
width:80%;
 +
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/modeling/zf21-16-2.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%'> The graph shows the relation between activation of the internal domain of CAR receptor and the nearly detected level of engineered exosomes.</span></p></div></html>
 +
<br><br>
 +
 +
2)We modeled the kinetics of Receptors Activated Solely by Synthetic Ligands (RASSLs) to explain production of engineered exosomes from MSC, but it’s concluded that it wasn’t efficient as there’s low detected signal of exosomes production as its internal domain transcription factor activates a lot of pathways so it will not be specific to activate our internal circuit.
 +
<html><div align="center"style="border:solid #17252A; width:100%;float:center;"><img style="                              max-width:850px;
 +
width:80%;
 +
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/modeling/zf21-16-3.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%'> The graph shows the relation between activation of the internal domain of RASSLs receptor and the low detected level of engineered exosomes.</span></p></div></html>
 +
<br><br>
 +
3)We modeled the kinetics of antibody scaffold receptor ( Example for it third generation of CAR receptors ) to explain production of engineered exosomes from MSC, but it’s concluded that it wasn’t efficient as there is no signal of exosomes production as its internal domain couldn’t be modified to be used in MSC .
 +
 +
<html><div align="center"style="border:solid #17252A; width:100%;float:center;"><img style="                              max-width:850px;
 +
width:80%;
 +
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/modeling/zf21-16-4.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%'> The graph shows the relation between activation of the internal domain of  antibody scaffold  receptors and the nearly detected level of engineered exosomes.</span></p></div></html>
 +
<br><br>
 +
4)We modeled the kinetics of syn-notch receptor  to explain production of engineered exosomes from MSC, it’s concluded that it is efficient as there’s a high signal of exosomes production as its internal domain can be modified to be used in MSC .
 +
 +
<html><div align="center"style="border:solid #17252A; width:100%;float:center;"><img style="                              max-width:850px;
 +
width:80%;
 +
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/modeling/zf21-16-5.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%'> The graph represents the relation between the activation of the internal domain of the syn-noth (represented as red line) and production of exosomes with specific cargo (represented as blue line) as the production of the engineered exosomes is initiated once the internal domain is activated.</span></p></div></html>
 +
==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.
 +
<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%'>
 +
 +
</span></p></div></html>
 +
<br><br><br><br>
 +
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>
 +
<br><br>
 +
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)
 +
<br>
 +
<html><div align="center"style="border:solid #17252A; width:75%;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/parts/vector-1.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>
 +
 +
==References==
 +
Nagano, M., Hoshino, D., Koshiba, S., Shuo, T., Koshikawa, N., Tomizawa, T., Hayashi, F., Tochio, N., Harada, T., Akizawa, T., Watanabe, S., Handa, N., Shirouzu, M., Kigawa, T., Yokoyama, S., & Seiki, M. (2011). ZF21 protein, a regulator of the disassembly of focal adhesions and cancer metastasis, contains a novel noncanonical pleckstrin homology domain. The Journal of biological chemistry, 286(36), 31598–31609. https://doi.org/10.1074/jbc.M110.199430
 +
<br><br><br>
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===

Latest revision as of 14:02, 12 October 2023


ZF21.16


Part Description

Zinc finger proteins are considered one of the most unique and huge collections of proteins that are variable in their biological function and structure. In our condition this zinc finger protein recognizes a specific DNA sequence thus ZF21.16 can be combined with any other protein as transcription factor to direct its activity toward the target sequences that are located near the gene of interest.

Usage

We used this part in our design to form the internal domain of our Syn-Notch receptor (BBa_K4586022). This internal domain is released after receptor activation and guided by the ZF21.16 to the target promoter that regulates the levels of expression of our therapeutic agent 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

The study used ZF21 antibody for detection of ZF21 by Western blot at 1 µg/mL. For immunofluorescence, start at 20 µg/mL.

(A)western blot analysis of ZF21 in 3T3 cell tissue lysate with ZF 21 antibodies in abscence of blocking peptide while (B) the same western blot analysis in presence of blocking peptide

charactrization of mathematical modeling

We are using mathematical modeling to simulate the effect of activation on the internal domain on the level of expression of our engineered exosomes so on activation of Zinc finger proteins (ZF21.16) which represent a part of the internal domain that leads to stimulation of the whole internal domain.

The graph shows the relation between activation of the internal domain of synthetic notch and increased level of engineered exosomes.



comparison between the types of internal domains of different receptors based on their ability for production of exosomes.
1)We modeled the kinetics of chimeric antigen receptor (CAR) to explain production of engineered exosomes from MSC, but it’s concluded that it wasn’t efficient as there’s nearly detected signal of exosomes production as its internal domain couldn’t be modified to be used in MSC

The graph shows the relation between activation of the internal domain of CAR receptor and the nearly detected level of engineered exosomes.



2)We modeled the kinetics of Receptors Activated Solely by Synthetic Ligands (RASSLs) to explain production of engineered exosomes from MSC, but it’s concluded that it wasn’t efficient as there’s low detected signal of exosomes production as its internal domain transcription factor activates a lot of pathways so it will not be specific to activate our internal circuit.

The graph shows the relation between activation of the internal domain of RASSLs receptor and the low detected level of engineered exosomes.



3)We modeled the kinetics of antibody scaffold receptor ( Example for it third generation of CAR receptors ) to explain production of engineered exosomes from MSC, but it’s concluded that it wasn’t efficient as there is no signal of exosomes production as its internal domain couldn’t be modified to be used in MSC .

The graph shows the relation between activation of the internal domain of antibody scaffold receptors and the nearly detected level of engineered exosomes.



4)We modeled the kinetics of syn-notch receptor to explain production of engineered exosomes from MSC, it’s concluded that it is efficient as there’s a high signal of exosomes production as its internal domain can be modified to be used in MSC .

The graph represents the relation between the activation of the internal domain of the syn-noth (represented as red line) and production of exosomes with specific cargo (represented as blue line) as the production of the engineered exosomes is initiated once the internal domain is activated.

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

Nagano, M., Hoshino, D., Koshiba, S., Shuo, T., Koshikawa, N., Tomizawa, T., Hayashi, F., Tochio, N., Harada, T., Akizawa, T., Watanabe, S., Handa, N., Shirouzu, M., Kigawa, T., Yokoyama, S., & Seiki, M. (2011). ZF21 protein, a regulator of the disassembly of focal adhesions and cancer metastasis, contains a novel noncanonical pleckstrin homology domain. The Journal of biological chemistry, 286(36), 31598–31609. https://doi.org/10.1074/jbc.M110.199430


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]