Revision as of 17:30, 3 October 2011

HokkaidoU Japan

iGEM 2011 Team of Hokkaido University

GSK tag

GSK tag was constructed by Julie Torruellas Garcia et al. It is phosphorylated only in eucaryotic cells. So, we can figure out if protein was injected in eucaryotic cells by detecting phosphorylated GSK tag. GSK tag is derived from first 13aa of GSK-3β. Because of it's small size, the interference in tagged protein should be at minimum. We removed present Spe I site in the sequence by silent mutation.

It was reported that GSK tag can be added to N terminus, C terminus and anywhere in between of the protein. T3S signal should be on N terminus, we inserted GSK tag between T3S signal and Bsa I cloning site.

Investigation of T3SS-injectable proteins

Here we will discuss the structure of proteins which are injected and which are not. We tried eight different proteins: mnt repressor, Gal4, RFP, GFP, Cre DNA recombinase, (CCR5) transmembrane, LacI and Luciferase. All were chosen from biobrick distribution which shows their significant importance for iGEM.

Our main concern was not with the size the protein but its stability. Previous research show that proteins like Zinc-Finger are were stable and couldn't be injected. Stability prevents unfolding by T3SS chaperons. Our asortment includes Gal4 which is representative of stable proteins.

Name	Registry	2011 distribution	length (bp)	total molecular weight (kDa)
Name	Discription
mnt repressor	[http://partsregistry.org/Part:BBa_C0072 BBa_C0072]	1-12L	288	42.1
mnt repressor	Discription
Gal4 DNA binding domain	[http://partsregistry.org/Part:BBa_K105007 BBa_K105007]	3-9I	438	47.6
Gal4 DNA binding domain	Discription
RFP	[http://partsregistry.org/Part:BBa_J06504 BBa_J06504]	1-13F	714	57.7
RFP	Discription
GFP	[http://partsregistry.org/Part:BBa_E0040 BBa_E0040]	1-14K	720	57.9
GFP	Discription
Cre DNA recombinase	[http://partsregistry.org/Part:BBa_J61047 BBa_J61047]	1-5D	1037	69.6
Cre DNA recombinase	Discription
CCR5	[http://partsregistry.org/Part:BBa_I712002 BBa_I712002]	2-3D	1059	70.4
CCR5	Discription
LacI	[http://partsregistry.org/Part:BBa_I732100 BBa_I732100]	2-10E	1086	71.4
LacI	Discription
Luciferase	[http://partsregistry.org/Part:BBa_I712019 BBa_I712019]	1-10H	1653	92.1
Luciferase	Discription

Team:HokkaidoU Japan/Project/GSK

From 2011.igem.org

Revision as of 17:30, 3 October 2011

HokkaidoU Japan

Contents

GSK tag

Investigation of T3SS-injectable proteins

@@ Line 27: / Line 27: @@
 ==GSK tag==
+GSK tag was constructed by Julie Torruellas Garcia et al. It is phosphorylated only in eucaryotic cells. So, we can figure out if protein was injected in eucaryotic cells by detecting phosphorylated GSK tag. GSK tag is derived from first 13aa of GSK-3β. Because of it's small size, the interference in tagged protein should be at minimum. We removed present Spe I site in the sequence by silent mutation.
+It was reported that GSK tag can be added to N terminus, C terminus and anywhere in between of the protein. T3S signal should be on N terminus, we inserted GSK tag between T3S signal and Bsa I cloning site.
+===Investigation of T3SS-injectable proteins===
+Here we will discuss the structure of proteins which are injected and which are not. We tried eight different proteins: mnt repressor, Gal4, RFP, GFP, Cre DNA recombinase, (CCR5) transmembrane, LacI and Luciferase. All were chosen from biobrick distribution which shows their significant importance for iGEM.
+Our main concern was not with the size the protein but its stability. Previous research show that proteins like Zinc-Finger are were stable and couldn't be injected. Stability prevents unfolding by T3SS chaperons. Our asortment includes Gal4 which is representative of stable proteins.