Part:BBa_K2794006
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 | [http://2018.igem.org/Team:SHSU_China 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.
Figure 1: pRK7-N-FLAG-WW Tag3-Vhb (PNCV)
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.
Figure 2: Results
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 139
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 157
Illegal NheI site found at 180 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 627
Illegal BamHI site found at 145 - 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 139
- 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 139
- 1000COMPATIBLE WITH RFC[1000]
| None |