Coding

Part:BBa_K2794006

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


WW Domain 3-Vhb Fusion Protein

WW3 Tagged Vhb
Function Active Cargo Loading
Use in Mammalian cells
RFC standard Not Compatible Due to Assembly
Backbone pSB1C3
Submitted by SHSU_China

Overview

This part is a composite part WW3 Tagged Vhb. The tagged protein will be attracted by Ndfip1 located on the endosome surface and then ubiquitinated by NEDD4 attracted by Ndfip1. Then the ubiquitinated protein will be transported into late endosome through ESCRT pathway.

Design

SHSU_China designed the pRK7-N-Flag-WW Tag3-Vhb plasmid to express the fusion protein inside the cell. T--SHSU China--PNW3V.png

Figure 1: pRK7-N-FLAG-WW Tag3-Vhb (PNW3V)

Prove of Expression

Team SHSU_China used HEK 293T cells in their exosome experiments. After using sequencing to conform the sequence of Ndfip, 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 PNW3V plasmids inside HEK 293T cells. Exosome content shown pale fusion protein band around 30kda. 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 COD value increased but the difference is not as huge as PNCV transfected once. T--SHSU China--results3.jpg

Figure 2: Results

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 139
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 157
    Illegal NheI site found at 180
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 627
    Illegal BamHI site found at 145
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 139
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 139
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
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