Difference between revisions of "Part:BBa K2794006"

Revision as of 16:01, 17 October 2018

WW Domain 3-Vhb Fusion Protein

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