Difference between revisions of "Part:BBa K4586026"
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<partinfo>BBa_K4586026 short</partinfo>
 
<partinfo>BBa_K4586026 short</partinfo>
  
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==Description==
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This composite part coding for our loading system that is composed of two components: L7Ae, which is a RNA binding protein that have an affinity to C\D box or kink turn coupled present within the 3` end of our cargo.
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This RNA binding protein is conjugated to the C terminal end of CD63, which is a highly expressed transmembrane protein on exosomes surface.
  
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==Part Description==
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==Usage==
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This part is implemented in our design to mediate the loading of our therapeutic agent into the engineered exosomes thus by labeling our cargo with a C\D box in the 3` prime end in order to bind to the L7Ae and load the cargo in the form of RNA selectively into the exosomes that target the autoreactive B-cells as shown in figure 1. and figure 2.
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<html><div align="center"style="border:solid #17252A; width:100%;float:center;"><img style="                              max-width:850px;
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width:75%;
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height:auto;
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position: relative;
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top: 50%;
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left: 35%;
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transform: translate( -50%);
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padding-bottom:25px;
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padding-top:25px;
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"src="https://static.igem.wiki/teams/4586/wiki/parts/14-15-26.png
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">
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<p class=MsoNormal align=center style='text-align:left;border:none;width:98% ;justify-content:center;'><span
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lang=EN style='font-size:11.0pt;line-height:115%'>Figure 1: This figure illustrates the mechanism of loading our therapeutic agent in the form of mRNA selectively and efficiently into our engineered exosomes secreted form the MSCs,this loading is done through labeling the gene of interest with C\D box a hairpin structure IN THE 3` end this box have high affinity to the RNA binding protein L7Ae that is expressed on the internal surface of the engineered exosomes membrane conjugated to his tagged CD63 protein that is  a natural highly expressed transmembrane protein within the exosomes.
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  </span></p></div></html>
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<br><br><br><br><br>
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<html><div align="center"style="border:solid #17252A; width:100%;float:center;"><img style="                              max-width:850px;
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width:50%;
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height:auto;
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position: relative;
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top: 50%;
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left: 25%;
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transform: translate( -50%);
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padding-bottom:25px;
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padding-top:25px;
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"src="https://static.igem.wiki/teams/4586/wiki/parts/14-15-26-part-2.png
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">
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<p class=MsoNormal align=center style='text-align:left;border:none;width:98% ;justify-content:center;'><span
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lang=EN style='font-size:11.0pt;line-height:115%'>Figure 2: This figure shows the design of the biological circuit expressing our loading system on the exosomes membrane ,this system consists of two main component ,First the RNA binding protein L7Ae conjugated to the second component, which is CD3 a transmembrane protein that is naturally expressed on the exosomes membrane.
  
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This part is coding for our intracellular transcription module, which is formed of 3 elements: the first is zinc finger 21.16, which drives our transcription module to its promoter; the second is nuclear localization signal SV40, which mediates the entrance of the transcription unit into the nucleus; the previous two elements are linked through a G4S linker in between; and the third is    VP64, which is a potent transcription factor.
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  </span></p></div></html>
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==Characterization By Mutational Landscape==
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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 (N120K) shows the highest epistatic fitness, while the lowest score was associated with the mutation (M115V,H134G,D145H).
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<html><div align="center"style="border:solid #17252A; width:80%;float:center;"><img style="                              max-width:850px;
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width:100%;
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height:auto;
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position: relative;
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top: 50%;
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left: 50%;
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transform: translate( -50%);
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padding-bottom:25px;
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padding-top:25px;
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"src="https://static.igem.wiki/teams/4586/wiki/parts-de/cd63-l7ae.png">
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<p class=MsoNormal align=center style='text-align:left;border:none;width:98% ;justify-content:center;'><span
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lang=EN style='font-size:11.0pt;line-height:115%'>Figure . An illustration of the effects of different mutations on the Epistatic Fitness of cd63-l7ae.  
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</span></p></div></html>
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==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 (P4) including CD63 an L7Ae, and then measuring the specific concentration of the running part using Real-Time PCR as shown in the following figure.
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<html><div align="center"style="border:solid #17252A; width:80%;float:center;"><img style="                              max-width:850px;
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width:100%;
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height:auto;
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position: relative;
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top: 50%;
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left: 50%;
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transform: translate( -50%);
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padding-bottom:25px;
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padding-top:25px;
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"src="https://static.igem.wiki/teams/4586/wiki/parts-experiments/pcr-ampli.png">
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<p class=MsoNormal align=center style='text-align:left;border:none;width:98% ;justify-content:center;'><span
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lang=EN style='font-size:11.0pt;line-height:115%'>
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</span></p></div></html>
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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 lane (P4).
 +
<html><div align="center"style="border:solid #17252A; width:80%;float:center;"><img style="                              max-width:850px;
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width:100%;
 +
height:auto;
 +
position: relative;
 +
top: 50%;
 +
left: 50%;
 +
transform: translate( -50%);
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padding-bottom:25px;
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padding-top:25px;
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"src="https://static.igem.wiki/teams/4586/wiki/parts-experiments/digestion-2.png">
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<p class=MsoNormal align=center style='text-align:left;border:none;width:98% ;justify-content:center;'><span
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lang=EN style='font-size:11.0pt;line-height:115%'>
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</span></p></div></html>
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<br><br>
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After the ligation step, we did a 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. This figure shows the Cell culture plate of transformed pCDNA vector 2 containing Loading system(CD63-L7Ae)-exosomal receptor(LAMP2B-anti-CD19)-connexin(CX43).
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<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/results/2.png">
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<p class=MsoNormal align=center style='text-align:left;border:none;width:98% ;justify-content:center;'><span
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lang=EN style='font-size:11.0pt;line-height:115%'>
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</span></p></div></html>
  
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==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.
 
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 14:13, 12 October 2023


Loading system (CD63-L7Ae)

Description

This composite part coding for our loading system that is composed of two components: L7Ae, which is a RNA binding protein that have an affinity to C\D box or kink turn coupled present within the 3` end of our cargo. This RNA binding protein is conjugated to the C terminal end of CD63, which is a highly expressed transmembrane protein on exosomes surface.

Usage

This part is implemented in our design to mediate the loading of our therapeutic agent into the engineered exosomes thus by labeling our cargo with a C\D box in the 3` prime end in order to bind to the L7Ae and load the cargo in the form of RNA selectively into the exosomes that target the autoreactive B-cells as shown in figure 1. and figure 2.

Figure 1: This figure illustrates the mechanism of loading our therapeutic agent in the form of mRNA selectively and efficiently into our engineered exosomes secreted form the MSCs,this loading is done through labeling the gene of interest with C\D box a hairpin structure IN THE 3` end this box have high affinity to the RNA binding protein L7Ae that is expressed on the internal surface of the engineered exosomes membrane conjugated to his tagged CD63 protein that is a natural highly expressed transmembrane protein within the exosomes.






Figure 2: This figure shows the design of the biological circuit expressing our loading system on the exosomes membrane ,this system consists of two main component ,First the RNA binding protein L7Ae conjugated to the second component, which is CD3 a transmembrane protein that is naturally expressed on the exosomes membrane.

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 (N120K) shows the highest epistatic fitness, while the lowest score was associated with the mutation (M115V,H134G,D145H).

Figure . An illustration of the effects of different mutations on the Epistatic Fitness of cd63-l7ae.

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 (P4) including CD63 an L7Ae, 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 (P4).



After the ligation step, we did a 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. This figure shows the Cell culture plate of transformed pCDNA vector 2 containing Loading system(CD63-L7Ae)-exosomal receptor(LAMP2B-anti-CD19)-connexin(CX43).


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]