Composite

Part:BBa_K5198105:Design

Designed by: Robert Kaptur   Group: iGEM24_Duke   (2024-08-23)


IFN-NanoLuc


HA-sfGFP composed of a hemagglutinins protein tag (BBa_K1150016) fused with sfGFP (BBa_K5198012), Superfolder green fluorescent protein. The HA-tag used is derived from the human influenza hemagglutinins protein, which aids the virus in targeting and entering a host cell[1]. HA-tag is not a signal sequence and therefore does not mediate export into specific subcellular compartments [2]. As the HA-tag is relatively small - 27 bp- it generally does not affect the traits of the protein attached. In the case of HA-sfGFP, this attached protein is superfolder GFP.

Description of the image

Figure 1. Fluorescence readings of sfGFP with different signal peptide fusions in HEK293T cell supernatant. The negative control (NC) is sfGFP fused with an HA tag, which does not exhibit extracellular secretion. Error bars represent the standard deviation (SD) of 4 biological replicates. The data was normalized by subtracting background fluorescence.


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
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 527


Design Notes

The HA-sfGFP composite protein was used in our secretion study as a null control. As the HA-tag is an epitope tag and not a signal sequence, this component of HA-sfGFP does not direct the secretion of the sfGFP to any specific space. The sfGFP protein served as our midsized protein (717 bp) in our experiment to see which signal sequences were optimal for the secretion of proteins of varying sizes


Source

References

[1] N. Nemanichvili et al., “Fluorescent trimeric hemagglutinins reveal multivalent receptor binding properties,” Journal of molecular biology, vol. 431, no. 4, p. 842, Feb. 2019, doi: 10.1016/j.jmb.2018.12.014. Available: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6397626/. [Accessed: Sep. 04, 2024]

[2] “HA-tag,” Wikipedia. Aug. 09, 2024. Available: https://en.wikipedia.org/w/index.php?title=HA-tag&oldid=1239490557. [Accessed: Sep. 04, 2024]