Difference between revisions of "Part:BBa K5499013"
 
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We constructed the IDS coding gene downstream of the strong CMV promoter, with the aim that, after transfecting HEK293T cells with the lentiviral expression vector containing this composite part, the cells would stably and abundantly express IDS.
 
We constructed the IDS coding gene downstream of the strong CMV promoter, with the aim that, after transfecting HEK293T cells with the lentiviral expression vector containing this composite part, the cells would stably and abundantly express IDS.
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K5499013 SequenceAndFeatures</partinfo>
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===Functional Parameters===
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<partinfo>BBa_K5499013 parameters</partinfo>
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===Characterization===
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===Usage and biology===
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We constructed this composite part into a lentiviral expression vector(Figure 1) and then transfected it into HEK293T cells.  
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The pLVX-Puro vector is a commonly used lentiviral expression vector designed to stably integrate foreign genes into the host cell genome, enabling long-term stable gene expression. The pLVX-Puro vector typically contains the human cytomegalovirus (CMV) promoter, which drives strong expression of the foreign gene. This vector also includes a puromycin resistance gene, allowing successfully transfected cells to survive in puromycin-containing selective media, facilitating the selection of cell lines stably expressing the target gene. Additionally, the vector contains a multiple cloning site (MCS), which allows easy insertion of foreign genes. <br>
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The IDS gene (NCBI ID: 3423) is located at chromosome region Xq28, spans 44 kb, and consists of 9 exons. The gene encodes a 550-amino-acid protein with a molecular weight of 61.8 kDa. Using homologous recombination, we successfully inserted the IDS gene into the pLVX-Puro vector (Figure 1) and constructed a stable cell line expressing this plasmid.
 
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<figure style="text-align:center;">
 
                 <img style="max-width:700px;" src="https://static.igem.wiki/teams/5499/registry-wang/first-genetation-plasmid.png" alt="control">
 
                 <img style="max-width:700px;" src="https://static.igem.wiki/teams/5499/registry-wang/first-genetation-plasmid.png" alt="control">
 
                 <figcaption><b>Figure 1:</b>  First Generation Plasmid Model Diagram</figcaption>
 
                 <figcaption><b>Figure 1:</b>  First Generation Plasmid Model Diagram</figcaption>
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===Verification===
 
The exosomes extracted from the culture medium were characterized using transmission electron microscopy (TEM) (Figure 2) to confirm their morphology and purity.
 
The exosomes extracted from the culture medium were characterized using transmission electron microscopy (TEM) (Figure 2) to confirm their morphology and purity.
 
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                 <img style="max-width:700px;" src="https://static.igem.wiki/teams/5499/registry-wang/tem-for-1st-exosomes.png" alt="control">
 
                 <img style="max-width:700px;" src="https://static.igem.wiki/teams/5499/registry-wang/tem-for-1st-exosomes.png" alt="control">
 
                 <figcaption><b>Figure 2:</b>  Exosomes Morphology Diagram by TEM</figcaption>
 
                 <figcaption><b>Figure 2:</b>  Exosomes Morphology Diagram by TEM</figcaption>
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Subsequently, we extracted total protein and exosomes from the cells and performed Western Blotting analysis.
 
Subsequently, we extracted total protein and exosomes from the cells and performed Western Blotting analysis.
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<figure style="text-align:center;">
 
<figure style="text-align:center;">
 
                 <img style="max-width:700px;" src="https://static.igem.wiki/teams/5499/registry-wang/wb-for-1st-genetation.png" alt="control">
 
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                 <figcaption><b>Figure 3:</b> Left Panel) Western Blotting Detection of Protein Content in cell Right Panel) Western Blotting Detection of Protein Content in Exosomes</figcaption>
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                 <figcaption><b>Figure 3:</b> Left Panel) Western Blotting Detection of Protein Content in cell <br>
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Right Panel) Western Blotting Detection of Protein Content in Exosomes</figcaption>
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K5499013 SequenceAndFeatures</partinfo>
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===In Conclusion===
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After the BBa_K5499013 sequence is integrated into the lentiviral expression vector, the transfected HEK293T cells will express large amounts of IDS. Even if IDS cannot be extensively sorted into exosomes for use in our project, future iGEM teams interested in the cellular synthesis of IDS can use it as a reference
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===Functional Parameters===
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<partinfo>BBa_K5499013 parameters</partinfo>
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Latest revision as of 12:34, 2 October 2024


Engineered exosome 1

We constructed the IDS coding gene downstream of the strong CMV promoter, with the aim that, after transfecting HEK293T cells with the lentiviral expression vector containing this composite part, the cells would stably and abundantly express IDS.

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]


Usage and biology

The pLVX-Puro vector is a commonly used lentiviral expression vector designed to stably integrate foreign genes into the host cell genome, enabling long-term stable gene expression. The pLVX-Puro vector typically contains the human cytomegalovirus (CMV) promoter, which drives strong expression of the foreign gene. This vector also includes a puromycin resistance gene, allowing successfully transfected cells to survive in puromycin-containing selective media, facilitating the selection of cell lines stably expressing the target gene. Additionally, the vector contains a multiple cloning site (MCS), which allows easy insertion of foreign genes.
The IDS gene (NCBI ID: 3423) is located at chromosome region Xq28, spans 44 kb, and consists of 9 exons. The gene encodes a 550-amino-acid protein with a molecular weight of 61.8 kDa. Using homologous recombination, we successfully inserted the IDS gene into the pLVX-Puro vector (Figure 1) and constructed a stable cell line expressing this plasmid.

control
Figure 1: First Generation Plasmid Model Diagram

Verification

The exosomes extracted from the culture medium were characterized using transmission electron microscopy (TEM) (Figure 2) to confirm their morphology and purity.

control
Figure 2: Exosomes Morphology Diagram by TEM
Subsequently, we extracted total protein and exosomes from the cells and performed Western Blotting analysis. As shown in Figure 3, the experimental group exhibited high levels of IDS expression in the cells compared to the blank control group (left panel). However, there was no significant difference in the expression levels of IDS in the exosomes between the experimental group and the blank control group (right panel).
control
Figure 3: Left Panel) Western Blotting Detection of Protein Content in cell
Right Panel) Western Blotting Detection of Protein Content in Exosomes

In Conclusion

After the BBa_K5499013 sequence is integrated into the lentiviral expression vector, the transfected HEK293T cells will express large amounts of IDS. Even if IDS cannot be extensively sorted into exosomes for use in our project, future iGEM teams interested in the cellular synthesis of IDS can use it as a reference