Difference between revisions of "Part:BBa K4586014"

 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K4586014 short</partinfo>
 
<partinfo>BBa_K4586014 short</partinfo>
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==Part Description==
 +
This part is the cell surface protein, which is a member of the tetraspanin family. It has numerous functions, such as being a marker for multivesicular bodies, quantifying extracellular vesicles, and can also be fused with other proteins to combine functional devices in the exosomal membrane.
  
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CD63 is a member of tetraspanin superfamily which is a hall mark natural protein highly expressed on the external surface of the exosomes.
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==Usage==
 +
We implemented this part in our design to form our loading system for the therapeutic agent to be loaded into exosomes. This loading system is composed of three elements: First, CD63, which is a transmembrane protein naturally present and highly expressed on the external surface of the exosome membrane, second L7Ae, which is a RNA-binding protein conjugated to the internal domain of the CD63 protein, in order to bind to the third element of the loading system present in the 3` end of our cargo, which is the C\D box or kink turn 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.
  
 +
  </span></p></div></html>
 +
==Literature Characterization==
 +
PCR was used to amplify the fragment of the outside membrane of SjCD63. The resulting cDNA was cloned into the pET-28a vector to produce the recombinant protein. The recombinant plasmids were then transformed into E. coli BL21 competent cells.
 +
The His-tagged CD63 recombinant protein was expressed in E. coli and purified using a Ni column
 +
<html><div align="center"style="border:solid #17252A; width:50%;float:center;"><img style="                              max-width:850px;
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width:75%;
 +
height:auto;
 +
position: relative;
 +
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/literature-characterisation-parts/cd63.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%'>SDS-PAGE of the purified recombinant protein showed a single band of the expected size, indicating that the purified CD63 protein was of high purity . The purified recombinant SjCD63 was further confirmed by mass spectrometry. In summary , a combination of PCR, cloning, expression, and purification techniques was used to produce a high-purity recombinant SjCD63 protein.
  
 +
</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 (D230N) shows the highest epistatic fitness, while the lowest score was associated with the mutation (V96S).
 +
<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.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.
 +
</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 (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.
 +
<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;
 +
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">
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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 insert parts.
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This plasmid contains Loading system(CD63-L7Ae)-exosomal receptor(LAMP2B-anti-CD19)-connexin(CX43)
 +
<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">
 +
<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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==References==
 +
Wang, L., Giri, B. R., Chen, Y., Xia, T., Liu, J., Li, H., ... & Cheng, G. (2018). Molecular characterization, expression profile, and preliminary evaluation of diagnostic potential of CD63 in Schistosoma japonicum. Parasitology research, 117, 3625-3631.
 
<!-- 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 14:01, 12 October 2023


CD63

Part Description

This part is the cell surface protein, which is a member of the tetraspanin family. It has numerous functions, such as being a marker for multivesicular bodies, quantifying extracellular vesicles, and can also be fused with other proteins to combine functional devices in the exosomal membrane.

Usage

We implemented this part in our design to form our loading system for the therapeutic agent to be loaded into exosomes. This loading system is composed of three elements: First, CD63, which is a transmembrane protein naturally present and highly expressed on the external surface of the exosome membrane, second L7Ae, which is a RNA-binding protein conjugated to the internal domain of the CD63 protein, in order to bind to the third element of the loading system present in the 3` end of our cargo, which is the C\D box or kink turn 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.

Literature Characterization

PCR was used to amplify the fragment of the outside membrane of SjCD63. The resulting cDNA was cloned into the pET-28a vector to produce the recombinant protein. The recombinant plasmids were then transformed into E. coli BL21 competent cells. The His-tagged CD63 recombinant protein was expressed in E. coli and purified using a Ni column

SDS-PAGE of the purified recombinant protein showed a single band of the expected size, indicating that the purified CD63 protein was of high purity . The purified recombinant SjCD63 was further confirmed by mass spectrometry. In summary , a combination of PCR, cloning, expression, and purification techniques was used to produce a high-purity recombinant SjCD63 protein.

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 (D230N) shows the highest epistatic fitness, while the lowest score was associated with the mutation (V96S).

Figure . An illustration of the effects of different mutations on the Epistatic Fitness of CD63.

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 insert parts. This plasmid contains Loading system(CD63-L7Ae)-exosomal receptor(LAMP2B-anti-CD19)-connexin(CX43)

References

Wang, L., Giri, B. R., Chen, Y., Xia, T., Liu, J., Li, H., ... & Cheng, G. (2018). Molecular characterization, expression profile, and preliminary evaluation of diagnostic potential of CD63 in Schistosoma japonicum. Parasitology research, 117, 3625-3631. 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]