Difference between revisions of "Part:BBa K4586009"
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lang=EN style='font-size:11.0pt;line-height:115%'>To confirm that iRGD-Lamp2b was successfully transfected into the imDCs, the amounts of iRGD-Lamp2b mRNA was measured 36 hours after transfection using RT-PCR.The results showed that the transfected imDCs expressed high levels of iRGD-Lamp2b mRNA, compared to the untransfected imDCs. This confirmed that the transfected imDCs were producing iRGD-positive exosomes. | lang=EN style='font-size:11.0pt;line-height:115%'>To confirm that iRGD-Lamp2b was successfully transfected into the imDCs, the amounts of iRGD-Lamp2b mRNA was measured 36 hours after transfection using RT-PCR.The results showed that the transfected imDCs expressed high levels of iRGD-Lamp2b mRNA, compared to the untransfected imDCs. This confirmed that the transfected imDCs were producing iRGD-positive exosomes. | ||
| + | </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 lane (p5) including lamb2p an anti-CD19 , and then we measured 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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| + | "src="https://static.igem.wiki/teams/4586/wiki/parts-experiments/pcr-ampli.png"> | ||
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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(P5). | ||
| + | <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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| + | 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) | ||
| + | <html><div align="center"style="border:solid #17252A; width:80%;float:center;"><img style=" max-width:850px; | ||
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Latest revision as of 14:06, 12 October 2023
lamp2b
Part Description
This part is the gene coding for a member of a family of membrane glycoproteins called Lysosome-associated membrane protein 2 (LAMP2), through modification of the outer membrane domain of the lamp2b. Our target protein can be delivered to the exosome's surface using Lamp 2b.
Usage
This part is used to express citrullinated vimentin on the exosomal surface to increase the exosomes' affinity for the autoreactive B-cells, as this transmembrane protein is unique to the membrane of exosomes and lysosomes, so the likelihood of exosome and B-cell fusion has significantly increased as shown in figure 1.
Figure 1: This figure illustrates the construction of our engineered exosomes that express citrullinated vimentin on their membranes conjugated to a transmembrane protein known as lysosome-associated membrane glycoprotein 2b (lamp2b), which is unique to the membrane of exosomes.
Literature Characterization
The study used X-Gal staining of brain sections from double transgenic mice to show the effect of active Notch1 signaling in mice. The results suggest that the activation of the N1-Gal4VP16 reporter is consistent with the activity of the endogenous Notch1 receptor. This reporter mouse is a powerful tool for visualizing active Notch1 signaling in embryonic and post-natal development in vivo.
To confirm that iRGD-Lamp2b was successfully transfected into the imDCs, the amounts of iRGD-Lamp2b mRNA was measured 36 hours after transfection using RT-PCR.The results showed that the transfected imDCs expressed high levels of iRGD-Lamp2b mRNA, compared to the untransfected imDCs. This confirmed that the transfected imDCs were producing iRGD-positive exosomes.
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 lane (p5) including lamb2p an anti-CD19 , and then we measured 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(P5).
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
Tian, Y., Li, S., Song, J., Ji, T., Zhu, M., Anderson, G. J., ... & Nie, G. (2014). A doxorubicin delivery platform using engineered natural membrane vesicle exosomes for targeted tumor therapy. Biomaterials, 35(7), 2383-2390. Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]