Coding

Part:BBa_K2794005

Designed by: LUO YAN   Group: iGEM18_SHSU_China   (2018-10-08)


CD63-Vhb Fusion Protein

CD63-Vhb Fusion Protein
Function Membrane Protein Anchoring
Use in Mammalian cells
RFC standard Not Compatible Due to Assembly
Backbone pSB1C3
Submitted by SHSU_China

Overview

This part is a composite part CD63-Vhb Fusion Protein. Vhb is connected to the 3 prime of CD63 sequence/ C- terminus of CD63 Protein. After translation in mammalian cell, the fusion protein will first be transported to the endosome and then through inward budding and exocytosis be exported on the membrane of exosomes. Vhb is located inside the exosome.

Design

SHSU_China designed the pRK7-N-Flag-CD63-Vhb plasmid to express the fusion protein inside the cell. T--SHSU China--PNCV.png

Figure 1: pRK7-N-FLAG-CD63-Vhb (PNCV)

Prove of Expression

Team SHSU_China used HEK 293T cells in their exosome experiments. After using sequencing to conform the sequence of the fusion protein, 2ug of vecter is transfected to a 60mm plate using lipofectamine 2000. Exosomes are extracted from the 4ml culture media using total exosome isolation kit (from cell culture media) from Invitrogen. Cells and exosomes are lysed using Ripa.

Cell content western blotting first proved successful translation of PNCV plasmids inside HEK 293T cells. Exosome content shown successful result of fusion protein band around 40 kda. TEM result of sample shown that the protein doesn’t effect the shape or the concentration of exosomes. COD result compared to normal exosomes shown that the fusion protein contains heme group that increases the chemical demand of exosomes and thus proving the function of Vhb is not disturbed. Also, color difference can be seen between normal and fusion-protein containing exosomes. T--SHSU China--results2.jpg T--SHSU China--improve1.png.jpg

Figure 2: Results


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 712
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 730
    Illegal NheI site found at 753
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1200
    Illegal BamHI site found at 718
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 712
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 712
  • 1000
    COMPATIBLE WITH RFC[1000]


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