Difference between revisions of "Part:BBa K3113302"
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This plasmid codes for the coat protein of gag fused to a split luciferase fused to an RNA binding protein. This construct forms virus-like particles which can be detected via a split luciferase assay. Through the RNA binding protein, the vesicles can be loaded with RNA. | This plasmid codes for the coat protein of gag fused to a split luciferase fused to an RNA binding protein. This construct forms virus-like particles which can be detected via a split luciferase assay. Through the RNA binding protein, the vesicles can be loaded with RNA. | ||
| − | + | <h2>Usage</h2> | |
pCAG_GAG-HiBit-L7Ae construct is a Composite part that has been designed to achieve VLP formation inside HEK293T/MIN6-K8 cells and enable the transport of chosen RNA information inside vesicles through L7Ae-C/D box interaction (together with construct BBa_K3113301<ref>https://parts.igem.org/Part:BBa_K3113301</ref> and BBa_K3113303<ref>https://parts.igem.org/Part:BBa_K3113303</ref>). | pCAG_GAG-HiBit-L7Ae construct is a Composite part that has been designed to achieve VLP formation inside HEK293T/MIN6-K8 cells and enable the transport of chosen RNA information inside vesicles through L7Ae-C/D box interaction (together with construct BBa_K3113301<ref>https://parts.igem.org/Part:BBa_K3113301</ref> and BBa_K3113303<ref>https://parts.igem.org/Part:BBa_K3113303</ref>). | ||
| − | + | <h2>Biology</h2> | |
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<partinfo>BBa_K3113302 SequenceAndFeatures</partinfo> | <partinfo>BBa_K3113302 SequenceAndFeatures</partinfo> | ||
| − | + | <h2>Characterization</h2> | |
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| − | Dynamic Light Scattering (DLS) | + | <h3>Dynamic Light Scattering (DLS)</h3> |
To confirm the size of VLPs calculated with the TEM pictures, we performed an analysis with dynamic light scattering. | To confirm the size of VLPs calculated with the TEM pictures, we performed an analysis with dynamic light scattering. | ||
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| − | Split-luciferase bioluminescent assay: The HiBiT Assay | + | <h3>Split-luciferase bioluminescent assay: The HiBiT Assay</h3> |
To furtherly prove that the BioBrick Part we designed works as expected we performed a HiBiT split luciferase assay, which shows luminescent signal detected in formed VLPs. | To furtherly prove that the BioBrick Part we designed works as expected we performed a HiBiT split luciferase assay, which shows luminescent signal detected in formed VLPs. | ||
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</html> | </html> | ||
| − | Affinity Purification | + | <h3>Affinity Purification</h3> |
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| − | |||
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<html> | <html> | ||
| − | <p style="text-align:center;"><img src="https://2019.igem.org/wiki/images/ | + | <p style="text-align:center;"><img src="https://2019.igem.org/wiki/images/c/cc/T--Munich--Validation_Purification.png" alt="CD63“ width="400" height="360"></p> |
</html> | </html> | ||
| − | Western Blot | + | <h3>Western Blot</h3> |
| − | qPCR Analysis | + | <h3>qPCR Analysis</h3> |
Finally, the BioBrick Part we designed works as expected since chosen RNA information is successfully transported into VLPs through L7Ae-C/D box interaction. This has been proven through qPCR analysis of VLP content. | Finally, the BioBrick Part we designed works as expected since chosen RNA information is successfully transported into VLPs through L7Ae-C/D box interaction. This has been proven through qPCR analysis of VLP content. | ||
| + | <html> | ||
| + | <p style="text-align:center;"><img src="https://2019.igem.org/wiki/images/5/54/T--Munich--qPCR_HEK_CC.png" alt="CD63“ width="400" height="360"></p> | ||
| + | </html> | ||
===References=== | ===References=== | ||
Revision as of 19:29, 20 October 2019
pCAG_Gag-HiBiT-L7Ae
This plasmid codes for the coat protein of gag fused to a split luciferase fused to an RNA binding protein. This construct forms virus-like particles which can be detected via a split luciferase assay. Through the RNA binding protein, the vesicles can be loaded with RNA.
Usage
pCAG_GAG-HiBit-L7Ae construct is a Composite part that has been designed to achieve VLP formation inside HEK293T/MIN6-K8 cells and enable the transport of chosen RNA information inside vesicles through L7Ae-C/D box interaction (together with construct BBa_K3113301[1] and BBa_K3113303[2]).
Biology
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 3019
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 2319
Characterization
Transmission Electron Microscopy (TEM)
Evidently, VLP formation from cells has been observed by transmission electron microscopy (TEM), proving the presence of the construct as the cause of VLP formation. GAG protein, encoded on the construct, is the main structural part of vesicle assembly.
Dynamic Light Scattering (DLS)
To confirm the size of VLPs calculated with the TEM pictures, we performed an analysis with dynamic light scattering.
Split-luciferase bioluminescent assay: The HiBiT Assay
To furtherly prove that the BioBrick Part we designed works as expected we performed a HiBiT split luciferase assay, which shows luminescent signal detected in formed VLPs.
Affinity Purification
Western Blot
qPCR Analysis
Finally, the BioBrick Part we designed works as expected since chosen RNA information is successfully transported into VLPs through L7Ae-C/D box interaction. This has been proven through qPCR analysis of VLP content.
