Difference between revisions of "Part:BBa K2794003"

Revision as of 14:21, 17 October 2018

WW Domain 3

WW Tag 3
Function	Active Cargo Loading for Exosomes
Use in	Mammalian cells
RFC standard	RFC 10 compatible
Backbone	pSB1C3
Submitted by	[http://2018.igem.org/Team:SHSU_China SHSU_China]

Overview

WW domain is a 40 amino acid length domain that can primarily bind to PPxY motif. It was first discovered on NEDD4 protein, enabling NEDD4 to bind with Ndfip, thus initiating ubquitination. Soon the interaction between WW domain and PPxY motif was found in many irrelevant signaling pathway including viral gag proteins, interleukin receptors and several serine/threonine kinases , indicating a universal significance. It’s worth to mention that WW domain on NEDD4 specifically can bind to an in vitro PY motif, which permits a more flexible way for experiments.

In experiments, in order to obtain the best binding efficiency with PPxY motif, Team SHSU_China chose the third and fourth from all 4 WW domains on NEDD4 to continue.

Design

Team SHSU_China used sequence of WW domain 3 from Human Nedd4 sequence and added HindIII and XbaI when designing the sequence. The following primers are used to sequence the Tags:

WW Tag-Forward:CCGGAATTCCCCAAGCTTCGTCGTGCCT

WW3-Reverse:TGCACTGCAGGCTCTAGACGGAATTTTCAGACGCGGATCTT

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 3: Results

Sequence and Features

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
COMPATIBLE WITH RFC[1000]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K2794003 short</partinfo>
-WW Domain 3
+{| style="color:black" cellpadding="6" cellspacing="1" border="2" align="right"
+! colspan="2" style="background:#A31F34;"|<span style="color:#ffffff">WW Tag 3</span>
+|-
+|'''Function'''
+|Active Cargo Loading for Exosomes
+|-
+|'''Use in'''
+|Mammalian cells
+|-
+|'''RFC standard'''
+|RFC 10 compatible
+|-
+|'''Backbone'''
+|pSB1C3<br>
+|-
+|'''Submitted by'''
+|[http://2018.igem.org/Team:SHSU_China SHSU_China]
+|}
-<!-- Add more about the biology of this part here
+==Overview==
-===Usage and Biology===
+WW domain is a 40 amino acid length domain that can primarily bind to PPxY motif. It was first discovered on NEDD4 protein, enabling NEDD4 to bind with Ndfip, thus initiating ubquitination. Soon the interaction between WW domain and PPxY motif was found in many irrelevant signaling pathway including viral gag proteins, interleukin receptors and several serine/threonine kinases , indicating a universal significance. It’s worth to mention that WW domain on NEDD4 specifically can bind to an in vitro PY motif, which permits a more flexible way for experiments.
+In experiments, in order to obtain the best binding efficiency with PPxY motif, Team SHSU_China chose the third and fourth from all 4 WW domains on NEDD4 to continue.
+==Design==
+Team SHSU_China used sequence of WW domain 3 from Human Nedd4 sequence and added HindIII and XbaI when designing the sequence. The following primers are used to sequence the Tags:
+'''WW Tag-Forward:'''CCGGAATTCCCCAAGCTTCGTCGTGCCT
+'''WW3-Reverse:'''TGCACTGCAGGCTCTAGACGGAATTTTCAGACGCGGATCTT
+==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.
+[[File:T--SHSU_China--results3.jpg|600px|]]
+'''Figure 3: Results'''
 <!-- -->