Part:BBa_K3113302

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 and BBa_K3113303).

Biology

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 3019
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE 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

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.

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.