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To furtherly prove the BioBrick Part we designed works as expected we performed a HiBiT split luciferase assay, which shows luminescent signal detected in formed VLPs. The following graph shows the amount of expressed Gag-HiBiT in cells compared to the amount measured in the supernatant. With this assay, we could show that 45 percent of Gag is exported to the supernatant in the form of vesicles.
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                     <figcaption class="figure-caption"><b>Figure 2:</b> Total Gag levels and export in VLPs in HEK393T cells.   
 
                     <figcaption class="figure-caption"><b>Figure 2:</b> Total Gag levels and export in VLPs in HEK393T cells.   
 
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              To furtherly prove the BioBrick Part we designed works as expected we performed a HiBiT split luciferase assay, which shows luminescent signal detected in formed VLPs. The following graph shows the amount of expressed Gag-HiBiT in cells compared to the amount measured in the supernatant. With this assay, we could show that 45 percent of Gag is exported to the supernatant in the form of vesicles.
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Revision as of 13:32, 21 October 2019


Gag HIV-1

This sequence codes for the coat protein of the human immunodeficiency virus, which mediates the essential events in virion assembly, including binding the plasma membrane, making the protein-protein interactions necessary to create spherical particles.


Usage

For ALiVE, two vesicles were tested. One kind of vesicle is based on the Gag-p24 capsid protein of HIV. We used this protein fused together with RNA binding proteins to build and load virus-like vesicles for non-invasive cell monitoring.


Biologie

Gag-p24 is the group-specific antigen from the lentivirus HIV. It assembles at the plasma membrane and leads to the budding of vesicles.[1]


Characterization

Protein Structure

A generic square placeholder image with rounded corners in a figure.
The structure of the viral Gag polyprotein.


HiBiT

To furtherly prove the BioBrick Part we designed works as expected we performed a HiBiT split luciferase assay, which shows luminescent signal detected in formed VLPs. The following graph shows the amount of expressed Gag-HiBiT in cells compared to the amount measured in the supernatant. With this assay, we could show that 45 percent of Gag is exported to the supernatant in the form of vesicles.

A generic square placeholder image with rounded corners in a figure.
Figure 2: Total Gag levels and export in VLPs in HEK393T cells.

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 1307
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 607


References

  1. ↑ HIV-1 Gag: a Molecular Machine Driving Viral Particle Assembly and Release Heinrich G. Göttlinger Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, and Department of Pathology, Harvard Medical School, Boston