Difference between revisions of "Part:BBa K5499010"

 
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<partinfo>BBa_K5499010 short</partinfo>
 
<partinfo>BBa_K5499010 short</partinfo>
  
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This sequence enables stable expression of an exosome containing the LAMP2A protein after transfecting HEK293T cells. Meanwhile, TAT will be connected to LAMP2A and displayed on the outer surface of the exosome, allowing the exosome to effectively penetrate biological barriers such as the blood-brain barrier, thereby creating an ideal drug delivery vehicle.
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To efficiently deliver the IDS protein and penetrate the blood-brain barrier, we designed a novel exosome vector based on research by João Vasco Ferreira et al.[1].
  
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===Usage and Biology===
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===Characterization===
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We added the Exosignal containing KFERQ pentapeptide motif to the C-terminus of the IDS protein, which is known to enhance exosome loading and release efficiency via a LAMP2a-mediated pathway(Figure 1).
  
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First, we performed a double digestion of the pLVX-Puro vector, linearizing it with EcoR I and Xba I. After gel electrophoresis and DNA purification, we obtained the linearized plasmid vector. We then amplified the DNA fragment containing the KFERQ pentapeptide motif from an existing IDS plasmid in the lab. Using homologous recombination, we ligated the linearized vector with the target fragment. The recombinant product was transformed into DH5α competent cells for culture. After antibiotic selection, we successfully obtained positive clones, and the correctness of the construction was confirmed by PCR and sequencing (Figure 1).
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<html>
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<figure style="text-align:center;">
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                <img style="max-width:700px;" src="https://static.igem.wiki/teams/5499/registry-wang/ids-exosignal.png" alt="control">
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                <figcaption><b>Figure 1:</b>  IDS-ExoSignal fragment: 1816 bp</figcaption>
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</figure>
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We constructed fragment part into a lentiviral expression vector(Figure 2) and then transfected it into HEK293T cells.
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<html>
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<figure style="text-align:center;">
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                <img style="max-width:700px;" src="https://static.igem.wiki/teams/5499/registry-wang/second-generation-plasmid.png" alt="control">
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                <figcaption><b>Figure 2:</b>  Sencond Generation Plasmid Model Diagram</figcaption>
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</figure>
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The exosomes extracted from the culture medium were characterized using transmission electron microscopy (TEM) (Figure 3) to confirm their morphology and purity.
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<html>
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<figure style="text-align:center;">
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                <img style="max-width:700px;" src="https://static.igem.wiki/teams/5499/registry-wang/tem-for-2nd-exosomes.png" alt="control">
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                <figcaption><b>Figure 3:</b>  Exosome Morphology Diagram by TEM</figcaption>
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</figure>
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We labeled the collected exosomes using DiI dye. A dye dilution was prepared, followed by incubation at room temperature in the dark. The labeled exosomes were then purified by ultrafiltration, resulting in a labeled exosome suspension.
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Western blot analysis confirmed a significant increase in IDS protein expression in the stable cell line, particularly in exosomes, where the IDS expression level was several times higher than that of the control group (Figure 4). This result demonstrates that we successfully constructed a cell line capable of high IDS expression in exosomes, meeting our design objectives.
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<html>
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<figure style="text-align:center;">
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                <img style="max-width:700px;" src="https://static.igem.wiki/teams/5499/registry-wang/wb-for-2nd.png" alt="control">
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                <figcaption><b>Figure 4:</b>  Western Blot Results Diagram</figcaption>
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</figure>
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We conducted exosome uptake experiments on U87MG cells using flow cytometry and confocal fluorescence microscopy. The results showed that the uptake efficiency of IDS ExoSignal small extracellular vesicles (sEVs) was similar to that of unmodified IDS sEVs (Figures 5 and 6), indicating that this iteration did not significantly enhance cellular uptake of the exosomes.
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<html>
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<figure style="text-align:center;">
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                <img style="max-width:700px;" src="https://static.igem.wiki/teams/5499/registry-wang/flow-for-2nd-in-single-chamber.png" alt="control">
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                <figcaption><b>Figure 5:</b>  Flow Cytometry Analysis of Exosome Uptake in Monolayer U87MG Cells</figcaption>
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</figure>
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<html>
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<figure style="text-align:center;">
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                <img style="max-width:700px;" src="https://static.igem.wiki/teams/5499/registry-wang/con-for-2nd-in-single-chamber-png.png" alt="control">
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                <figcaption><b>Figure 6:</b>  Confocal Fluorescence Imaging of Exosome Uptake in Monolayer U87MG Cells</figcaption>
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</figure>
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we successfully constructed an in vitro BBB model by co-culturing human brain microvascular endothelial cells (HCMEC/D3) with astrocytoma cells (U87MG)(Figure 7).
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<html>
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<figure style="text-align:center;">
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                <img style="max-width:700px;" src="https://static.igem.wiki/teams/5499/registry-wang/bbb-model.png" alt="control">
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                <figcaption><b>Figure 7:</b>  In Vitro Blood-Brain Barrier Model Diagram</figcaption>
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</figure>
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In the in vitro blood-brain barrier model, we found that both IDS sEVs and IDS ExoSignal sEVs exhibited low penetration efficiency and failed to effectively cross the BBB to reach U87MG cells in the lower chamber .By observing the two images (Figures 8 and 9), we can conclude that the lentiviral expression vector containing this composite part, after transfecting HEK293T cells, does not significantly enhance the engineered exosomes' ability to penetrate the blood-brain barrier.
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<html>
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<figure style="text-align:center;">
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                <img style="max-width:700px;" src="https://static.igem.wiki/teams/5499/registry-wang/flow-for-2nd-in-double-chamber-png.png" alt="control">
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                <figcaption><b>Figure 8:</b>  Flow Cytometry Analysis of Exosome Uptake in U87MG Cells in the Lower Chamber of an In Vitro BBB Model</figcaption>
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</figure>
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<html>
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<figure style="text-align:center;">
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                <img style="max-width:700px;" src="https://static.igem.wiki/teams/5499/registry-wang/con-for-2nd-in-double-chamber-png.png" alt="control">
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                <figcaption><b>Figure 9:</b>  Confocal Fluorescence Imaging of Exosome Uptake in U87MG Cells in the Lower Chamber of an In Vitro BBB Model</figcaption>
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</figure>
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Revision as of 05:22, 2 October 2024


Engineered exosome 2

To efficiently deliver the IDS protein and penetrate the blood-brain barrier, we designed a novel exosome vector based on research by João Vasco Ferreira et al.[1].

Characterization

We added the Exosignal containing KFERQ pentapeptide motif to the C-terminus of the IDS protein, which is known to enhance exosome loading and release efficiency via a LAMP2a-mediated pathway(Figure 1).

First, we performed a double digestion of the pLVX-Puro vector, linearizing it with EcoR I and Xba I. After gel electrophoresis and DNA purification, we obtained the linearized plasmid vector. We then amplified the DNA fragment containing the KFERQ pentapeptide motif from an existing IDS plasmid in the lab. Using homologous recombination, we ligated the linearized vector with the target fragment. The recombinant product was transformed into DH5α competent cells for culture. After antibiotic selection, we successfully obtained positive clones, and the correctness of the construction was confirmed by PCR and sequencing (Figure 1).

control
Figure 1: IDS-ExoSignal fragment: 1816 bp
We constructed fragment part into a lentiviral expression vector(Figure 2) and then transfected it into HEK293T cells.
control
Figure 2: Sencond Generation Plasmid Model Diagram
The exosomes extracted from the culture medium were characterized using transmission electron microscopy (TEM) (Figure 3) to confirm their morphology and purity.
control
Figure 3: Exosome Morphology Diagram by TEM
We labeled the collected exosomes using DiI dye. A dye dilution was prepared, followed by incubation at room temperature in the dark. The labeled exosomes were then purified by ultrafiltration, resulting in a labeled exosome suspension. Western blot analysis confirmed a significant increase in IDS protein expression in the stable cell line, particularly in exosomes, where the IDS expression level was several times higher than that of the control group (Figure 4). This result demonstrates that we successfully constructed a cell line capable of high IDS expression in exosomes, meeting our design objectives.
control
Figure 4: Western Blot Results Diagram
We conducted exosome uptake experiments on U87MG cells using flow cytometry and confocal fluorescence microscopy. The results showed that the uptake efficiency of IDS ExoSignal small extracellular vesicles (sEVs) was similar to that of unmodified IDS sEVs (Figures 5 and 6), indicating that this iteration did not significantly enhance cellular uptake of the exosomes.
control
Figure 5: Flow Cytometry Analysis of Exosome Uptake in Monolayer U87MG Cells
control
Figure 6: Confocal Fluorescence Imaging of Exosome Uptake in Monolayer U87MG Cells
we successfully constructed an in vitro BBB model by co-culturing human brain microvascular endothelial cells (HCMEC/D3) with astrocytoma cells (U87MG)(Figure 7).
control
Figure 7: In Vitro Blood-Brain Barrier Model Diagram
In the in vitro blood-brain barrier model, we found that both IDS sEVs and IDS ExoSignal sEVs exhibited low penetration efficiency and failed to effectively cross the BBB to reach U87MG cells in the lower chamber .By observing the two images (Figures 8 and 9), we can conclude that the lentiviral expression vector containing this composite part, after transfecting HEK293T cells, does not significantly enhance the engineered exosomes' ability to penetrate the blood-brain barrier.
control
Figure 8: Flow Cytometry Analysis of Exosome Uptake in U87MG Cells in the Lower Chamber of an In Vitro BBB Model
control
Figure 9: Confocal Fluorescence Imaging of Exosome Uptake in U87MG Cells in the Lower Chamber of an In Vitro BBB Model
Sequence and Features BBa_K5499010 SequenceAndFeatures