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<sup>[[#References|[1]]]</sup>. Ninth amino acid, serine, is phosphorylated in eukaryotic cells (Fig. 1). GSK tags phosphorylated state can be specifically detected by using phospho-specific antibodies. So it is effective method 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
 +
rm Spe I  :ATG AGT GGT CGC CCT CGC ACT ACA* AGT TTC GCT GAA AGT  
 +
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[1], C terminus[1] and anywhere in middle[2] of the protein. We opted to insert it between SlrP secretion tag and the protein we wanted to inject.
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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 located the tag between SlrP secretion tag and the protein fused to it.
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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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Non-phosphospecific antibodies can used for determination of total amount of expressed fusion protein within the assay.
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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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==Construction of GSK tagged T3SS-injectable proteins==
 +
Here we show a list of proteins which are to be tested for protein screening using GSK tag. We chose 8 different proteins (Table. 1). All are from iGEM 2011 biobrick distribution kit. As these parts are widely used in iGEM, characterising them would have a bigger impact compared to exotic ones.
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===Investigation of T3SS-injectable proteins===
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Our main concern is not with the size but the stability of proteins against unfolding by T3SS chaperone. Previous research indicate that proteins containing Zinc-Fingers are very stable and couldn't be injected. Proteins containing such stable structure is thought to resist unfolding by T3SS chaperon.
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Here we will discuss the structure of proteins which are injected and which are 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.
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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.
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We cloned these proteins into the Bsa I cloning site mentioned [[Team:HokkaidoU_Japan/Project/Backbone|here]].  
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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.  
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We have previously shown that GFP can be injected into eucaryotic cells by observation under confocal laser microscope. Thus we have a positive control.
{|class="protein" style="text-align:center;"
{|class="protein" style="text-align:center;"
|-
|-
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!rowspan="2"|Name
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!Name
|Registry
|Registry
|2011 distribution
|2011 distribution
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|total molecular weight (kDa)
|total molecular weight (kDa)
|-
|-
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|colspan="4" style="text-align:left;"|Discription
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!mnt repressor  
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|-
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!rowspan="2"|mnt repressor  
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|[http://partsregistry.org/Part:BBa_C0072 BBa_C0072]
|[http://partsregistry.org/Part:BBa_C0072 BBa_C0072]
|1-12L
|1-12L
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|42.1
|42.1
|-
|-
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|colspan="4" style="text-align:left;"|Discription
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!Gal4 DNA binding domain
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|-
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!rowspan="2"|Gal4 DNA binding domain
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|[http://partsregistry.org/Part:BBa_K105007 BBa_K105007]
|[http://partsregistry.org/Part:BBa_K105007 BBa_K105007]
|3-9I
|3-9I
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|47.6
|47.6
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|-
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|colspan="4" style="text-align:left;"|Discription
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!RFP
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|-
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!rowspan="2"|RFP
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|[http://partsregistry.org/Part:BBa_J06504 BBa_J06504]
|[http://partsregistry.org/Part:BBa_J06504 BBa_J06504]
|1-13F
|1-13F
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|57.7
|57.7
|-
|-
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|colspan="4" style="text-align:left;"|Discription
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!GFP
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|-
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!rowspan="2"|GFP
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|[http://partsregistry.org/Part:BBa_E0040 BBa_E0040]
|[http://partsregistry.org/Part:BBa_E0040 BBa_E0040]
|1-14K
|1-14K
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|57.9
|57.9
|-
|-
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|colspan="4" style="text-align:left;"|Discription
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!Cre DNA recombinase
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|-
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!rowspan="2"|Cre DNA recombinase
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|[http://partsregistry.org/Part:BBa_J61047 BBa_J61047]
|[http://partsregistry.org/Part:BBa_J61047 BBa_J61047]
|1-5D
|1-5D
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|69.6
|69.6
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|colspan="4" style="text-align:left;"|Discription
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!CCR5
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|-
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!rowspan="2"|CCR5
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|[http://partsregistry.org/Part:BBa_I712002 BBa_I712002]
|[http://partsregistry.org/Part:BBa_I712002 BBa_I712002]
|2-3D
|2-3D
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|70.4
|70.4
|-
|-
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|colspan="4" style="text-align:left;"|Discription
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!LacI
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|-
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!rowspan="2"|LacI
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|[http://partsregistry.org/Part:BBa_I732100 BBa_I732100]
|[http://partsregistry.org/Part:BBa_I732100 BBa_I732100]
|2-10E
|2-10E
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|71.4
|71.4
|-
|-
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|colspan="4" style="text-align:left;"|Discription
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!Luciferase
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|-
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!rowspan="2"|Luciferase
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|[http://partsregistry.org/Part:BBa_I712019 BBa_I712019]
|[http://partsregistry.org/Part:BBa_I712019 BBa_I712019]
|1-10H
|1-10H
|1653
|1653
|92.1
|92.1
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|-
 
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|colspan="4" style="text-align:left;"|Discription
 
|}
|}
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Table. 1 A list of several recommended proteins to be injected. Total molecular wight of each protein contains T3S signal and GSK tag domain.
 +
 +
However, of the time constraints we done only Pilot test which failed as a experiment for unknown causes so we don`t have a data to show.
 +
 +
To confirm the expression of the GSK tagged SlrP fusion proteins in E. coli  bacterial cell lysate must be analyzed by SDS-PAGE and immunoblotting with a GSK-3β(Cell　Signaling Technologies #9332 ) and a phosphospecific GSK-3β(Cell　Signaling Technologies #9336) antibody preparation.
=References=
=References=
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* 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]
* 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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{{Team:HokkaidoU_Japan/footer}}
{{Team:HokkaidoU_Japan/footer}}

Latest revision as of 12:55, 15 December 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). GSK tags phosphorylated state can be specifically detected by using phospho-specific antibodies. So it is effective method 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 located the tag between SlrP secretion tag and the protein fused to it.

Non-phosphospecific antibodies can used for determination of total amount of expressed fusion protein within the assay.

Construction of GSK tagged T3SS-injectable proteins

Here we show a list of proteins which are to be tested for protein screening using GSK tag. We chose 8 different proteins (Table. 1). All are from iGEM 2011 biobrick distribution kit. As these parts are widely used in iGEM, characterising them would have a bigger impact compared to exotic ones.

Our main concern is not with the size but the stability of proteins against unfolding by T3SS chaperone. Previous research indicate that proteins containing Zinc-Fingers are very stable and couldn't be injected. Proteins containing such stable structure is thought to resist unfolding by T3SS chaperon.

We cloned these proteins into the Bsa I cloning site mentioned here.

We have previously shown that GFP can be injected into eucaryotic cells by observation under confocal laser microscope. Thus we have a positive control.

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

Table. 1 A list of several recommended proteins to be injected. Total molecular wight of each protein contains T3S signal and GSK tag domain.

However, of the time constraints we done only Pilot test which failed as a experiment for unknown causes so we don`t have a data to show.

To confirm the expression of the GSK tagged SlrP fusion proteins in E. coli bacterial cell lysate must be analyzed by SDS-PAGE and immunoblotting with a GSK-3β(Cell　Signaling Technologies #9332 ) and a phosphospecific GSK-3β(Cell　Signaling Technologies #9336) antibody preparation.

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]
Retrieved from "http://2011.igem.org/Team:HokkaidoU_Japan/Project/GSK"