Team:HokkaidoU Japan/Project/GSK

From 2011.igem.org

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==GSK tag==
==GSK tag==
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Glycogen Synthase Kinase 3β is known to be phosphorylated by several enzymes in eukaryotic cells. We used first 13 amino acid as a tag (GSK tag) of injected fusion protein[1]. Ninth amino acid, serine, is phosphorylated in eukaryotic cells (Fig. 1). Phosphorylated serine in the polypeptide could be detected by using phsopho-specific antibodies which bind to only phosphorylated GSK tag. So it is effective to distinguish GSK tag fusion protein existing in eukaryotic cells from uninjected protein remaining ''E. coli''.
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Glycogen Synthase Kinase 3β is known to be phosphorylated by several enzymes in eukaryotic cell. We used first 13 amino acid as a tag (GSK tag)[1]. Ninth amino acid, serine is phosphorylated in eukaryotic cell(Fig). This phosphorylation state could be detected by using phsopho-specific antibodies which bind to only phosphorylated GSK tag. This way it is possible to distinguish whether GSK tag has been it eukaryotic cell. So you can see proteins which were injected into cell and which were not. This was a vital ingredient in our experiments.
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GSK tag was constructed by Julie Torruellas Garcia, Gregory V. Plano et al. We removed present Spe I site in the sequence by silent mutation.
GSK tag was constructed by Julie Torruellas Garcia, Gregory V. Plano et al. We removed present Spe I site in the sequence by silent mutation.
<pre>
<pre>
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Translation: M  S  G  R  P  R  T  T  S-p  F  A  E  S
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Fig. 1
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Original  :ATG AGT GGT CGC CCT CGC ACT ACT  AGT TTC GCT GAA AGT
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Translation: M  S  G  R  P  R  T  T  S-p  F  A  E  S
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rm Spe I  :ATG AGT GGT CGC CCT CGC ACT ACA* AGT TTC GCT GAA AGT  
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Original  :ATG AGT GGT CGC CCT CGC ACT ACT  AGT TTC GCT GAA AGT
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rm Spe I  :ATG AGT GGT CGC CCT CGC ACT ACA* AGT TTC GCT GAA AGT  
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Phosphorylated Serine is shown as S-p.
</pre>
</pre>
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Phosphorylated Serine is shown as S-p.
 
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GSK tag can be added to N terminus<sup>[[#References|[1]]]</sup>, C terminus<sup>[[#References|[1]]]</sup>, and anywhere in middle<sup>[[#References|[2]]]</sup>, of the protein. We opted to insert it between SlrP secretion tag and the protein we wanted to inject.
 
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Using non-phosphospecific antibodies it is possible to check the total amount of expressed protein with the tag. Comparing it with the injected protein you can determine the efficiency of the injection.
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GSK tag can be added to N terminus<sup>[[#References|[1]]]</sup>, C terminus<sup>[[#References|[1]]]</sup>, and anywhere in middle<sup>[[#References|[2]]]</sup>, of the protein. We alocated the tag between SlrP secretion tag and the protein we to be injected.
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By comparing the mass of the protein with GSK tag it is also possible to see if it had been modified in eucaryotic cell. It can be used alongside of TEV site and provide proof for successful TEV protease activity. An experiment we would like to try in the future.
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Non-phosphospecific antibodies for determination of total amount of expressed fusion protein with the tag we used.
==Investigation of T3SS-injectable proteins==
==Investigation of T3SS-injectable proteins==

Revision as of 18:47, 5 October 2011

Contents

GSK tag

Glycogen Synthase Kinase 3β is known to be phosphorylated by several enzymes in eukaryotic cells. We used first 13 amino acid as a tag (GSK tag) of injected fusion protein[1]. Ninth amino acid, serine, is phosphorylated in eukaryotic cells (Fig. 1). Phosphorylated serine in the polypeptide could be detected by using phsopho-specific antibodies which bind to only phosphorylated GSK tag. So it is effective to distinguish GSK tag fusion protein existing in eukaryotic cells from uninjected protein remaining E. coli.

GSK tag was constructed by Julie Torruellas Garcia, Gregory V. Plano et al. We removed present Spe I site in the sequence by silent mutation. 

Fig. 1
 Translation: M   S   G   R   P   R   T   T   S-p  F   A   E   S
 Original   :ATG AGT GGT CGC CCT CGC ACT ACT  AGT TTC GCT GAA AGT
 rm Spe I   :ATG AGT GGT CGC CCT CGC ACT ACA* AGT TTC GCT GAA AGT 
Phosphorylated Serine is shown as S-p.


GSK tag can be added to N terminus[1], C terminus[1], and anywhere in middle[2], of the protein. We alocated the tag between SlrP secretion tag and the protein we to be injected.

Non-phosphospecific antibodies for determination of total amount of expressed fusion protein with the tag we used.

Investigation of T3SS-injectable proteins

Here we will discuss about the structure of proteins which could be injected or not. We tried five different proteins: mnt repressor, RFP, GFP, Cre DNA recombinase, (CCR5) transmembrane, LacI and Luciferase. All were chosen from biobrick distribution. As these parts are widely used in iGEM studying them would have a bigger impact compared to exotic ones.

Our main concern was not with the size but the stability of proteins. Previous research show that proteins like Zinc-Fingers are very stable and couldn't be injected. Hight stability prevents unfolding by T3SS chaperons.

We showed that GFP can be injected into eucaryotic cells by confocal laser microscope imaging. Thus it can serve as a control. Next is RFG, a fluorescent protein but with different structure from GFP.


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

References

  • Julie Torruellas Garcia, Franco Ferracci, Michael W. Jackson,1 Sabrina S. Joseph, Isabelle Pattis, Lisa R. W. Plano, Wolfgang Fischer, and Gregory V. Plano. 2006. Measurement of Effector Protein Injection by Type III and Type IV Secretion Systems by Using a 13-Residue Phosphorylatable Glycogen Synthase Kinase Tag. Infect Immun.Vol.74:5645-57. [http://www.ncbi.nlm.nih.gov/pubmed/16988240 PubMed]
  • JWensheng Luo and Michael S. Donnenberg. 2011. Interactions and Predicted Host Membrane Topology of the Enteropathogenic Escherichia coli Translocator Protein EspB. J. Bacteriol.Vol.193:2972–80. [http://www.ncbi.nlm.nih.gov/pubmed/21498649 PubMed]
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