Difference between revisions of "Part:BBa K4586010"

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(→‎Experimental Characterization)
 
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==Usage==
 
==Usage==
−
This part acts to strengthen the duration and intensity of downstream signaling as target-ligand binding occurs. We implemented this in our system to improve the efficacy of our therapeutic agent by increasing the default level of exosome synthesis within our engineered MSC.
+
This part acts to strengthen the duration and intensity of downstream signaling as target-ligand binding occurs. We implemented this in our system to improve the efficacy of our therapeutic agent by increasing the default level of exosome synthesis within our engineered MSC 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/booster-gene.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 coding for booster genes(SDC4,STEAP3 and NadB) and their role in increasing the synthetic capacity of MSCs to secrete exosomes that carry our therapeutic agent represented in Cas12k/gBAFF-R
 +
  </span></p></div></html>
 +
==Literature Characterization==
 +
The study created a reporter construct by joining the C-terminus of CD63, one of the most used exosome markers, to nanoluc (nluc), a tiny and potent bioluminescence reporter10. After progressive centrifugation to eliminate masking signals12, luminescence in the cell-culture supernatant was measured. This reporter gene was co-transfected with plasmids expressing potential candidates for exosome production augmentation.
 +
<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/booster-genes.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 study found STEAP3 syndecan-4 (SDC4), and (NadB) as potential synthetic exosome production boosters. Combined expression of these genes significantly increased exosome production, and a tricistronic plasmid vector ( known as exosome production booster), which guarantees that transfected cells receive all boosted genes at a fixed ratio ,produced a 15-fold to 40-fold increase (depending on cell conditions) in the luminescence signal in the supernatant.
 +
</span></p></div></html>
 +
==Characterization By Mutational Landscape==
 +
In order to optimize the function of our parts, we've used the concept of Directed Evolution through applying different mutations and measuring the effects of these mutations on their evolutionary epistatic fitness.  As displayed in the chart below, the mutation (M169V) shows the highest epistatic fitness, while the lowest score was associated with the mutation (T144N).
 +
<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-de/sdc4.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 . An illustration of the effects of different mutations on the Epistatic Fitness of sdc4.
 +
</span></p></div></html>
 +
==charactrization by mathematical modeling==
 +
Presence of SDC4 as a booster gene has a role in increasing the concentration of the produced engineered exosomes so it plays an effective role to increase the efficacy of the therapeutic agent.
  
 +
We compared both condition of exosomes production when using booster genes and without it
 +
<br><br>
 +
(1)No booster genes with conditioned release
 +
<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/modeling/no-booster.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%'>This Represents the relation between the activation of the internal domain of the Syn-Notch (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>
 +
<br><br>
 +
(2)Booster gene with conditioned release
 +
<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/modeling/booster.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%'>This Represents the relation between the activation of the internal domain of the Syn-Notch (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 (P2) including STEAP3 and SDC4, 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 lane (P2).
 +
<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==
 +
Kojima, R., Bojar, D., Rizzi, G., Hamri, G. C. E., El-Baba, M. D., Saxena, P., ... & Fussenegger, M. (2018). Designer exosomes produced by implanted cells intracerebrally deliver therapeutic cargo for Parkinson’s disease treatment. Nature communications, 9(1), 1305.
 
<!-- 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 14:07, 12 October 2023


SDC4

Part Description

This part is an activator of signaling pathways to control cellular processes that arbitrate cell migration, proliferation, endocytosis, and mechano-transduction, as well as its ability to independently activate signaling pathways in response the target-ligand binding as well.

Usage

This part acts to strengthen the duration and intensity of downstream signaling as target-ligand binding occurs. We implemented this in our system to improve the efficacy of our therapeutic agent by increasing the default level of exosome synthesis within our engineered MSC as shown in figure 1.

Figure 1: This figure illustrates the design of our biological circuit coding for booster genes(SDC4,STEAP3 and NadB) and their role in increasing the synthetic capacity of MSCs to secrete exosomes that carry our therapeutic agent represented in Cas12k/gBAFF-R

Literature Characterization

The study created a reporter construct by joining the C-terminus of CD63, one of the most used exosome markers, to nanoluc (nluc), a tiny and potent bioluminescence reporter10. After progressive centrifugation to eliminate masking signals12, luminescence in the cell-culture supernatant was measured. This reporter gene was co-transfected with plasmids expressing potential candidates for exosome production augmentation.

The study found STEAP3 syndecan-4 (SDC4), and (NadB) as potential synthetic exosome production boosters. Combined expression of these genes significantly increased exosome production, and a tricistronic plasmid vector ( known as exosome production booster), which guarantees that transfected cells receive all boosted genes at a fixed ratio ,produced a 15-fold to 40-fold increase (depending on cell conditions) in the luminescence signal in the supernatant.

Characterization By Mutational Landscape

In order to optimize the function of our parts, we've used the concept of Directed Evolution through applying different mutations and measuring the effects of these mutations on their evolutionary epistatic fitness. As displayed in the chart below, the mutation (M169V) shows the highest epistatic fitness, while the lowest score was associated with the mutation (T144N).

Figure . An illustration of the effects of different mutations on the Epistatic Fitness of sdc4.

charactrization by mathematical modeling

Presence of SDC4 as a booster gene has a role in increasing the concentration of the produced engineered exosomes so it plays an effective role to increase the efficacy of the therapeutic agent.

We compared both condition of exosomes production when using booster genes and without it

(1)No booster genes with conditioned release

This Represents the relation between the activation of the internal domain of the Syn-Notch (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.



(2)Booster gene with conditioned release

This Represents the relation between the activation of the internal domain of the Syn-Notch (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 (P2) including STEAP3 and SDC4, 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 lane (P2).



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

Kojima, R., Bojar, D., Rizzi, G., Hamri, G. C. E., El-Baba, M. D., Saxena, P., ... & Fussenegger, M. (2018). Designer exosomes produced by implanted cells intracerebrally deliver therapeutic cargo for Parkinson’s disease treatment. Nature communications, 9(1), 1305. Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 201
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]