Team:HokkaidoU Japan/Project

From 2011.igem.org

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<div id="hokkaidou-right-content">
=Abstract=
=Abstract=
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Bacteria living around us evolved ways to effect their surrounding environment. This is done by using secretion systems. Type 3 Secretion System can inject whole protein molecules into targeted eukaryotic cells. During iGEM 2010 we showed that E. coli with a part of Salmonella genome library expresses T3SS. We thought this system can be applied to direct reprogramming of somatic cells among many other things.  
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Bacteria living around us evolved ways to effect their surrounding environment. Some bacteria can change its surroundings by injecting whole protein molecules into targeted eukaryotic cells through Type 3 secretion system (T3SS). During iGEM 2010 we showed that ''E. coli'' containing a part of ''Salmonella'' genome expresses T3SS. We thought this system can be applied to direct reprogramming of somatic cells from eukaryote.  
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This year we tried to make it more convenient. For this purpose we designed a plasmid backbone which can instantly produce ready-to-inject fusion proteins from ordinary biobrick part. Using it, we tried to further characterize this system by injecting characteristic proteins to see if they can be secreted.
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This year we tried to make it more convenient. For this purpose we designed a plasmid backbone which can instantly produce ready-to-inject fusion proteins from biobrick parts. Using it, we tried further characterization of this system by injecting various proteins to see if they can be secreted.
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=Introduction to T3SS=
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=What`s T3SS?=
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T3SS is a system of pathogenic gram-negative bacterium such as Salmonella, Yersinia and EPEC (entero pathogenic E. coli). Using this system bacteria can inject whole protein molecules through a syringe like organelle named T3S Apparatus. The target of this system is a eukaryotic cell. Naturally it is used to inject Virulence effector proteins. Last year, we showed that T3SS works in E. coli by injecting GFP into RK13 cells.
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T3SS is a system of pathogenic gram-negative bacterium such as ''Salmonella'', ''Yersinia'' and EPEC (entero pathogenic ''E. coli''). Using this system bacteria can inject whole protein molecules through a syringe like organelle named T3S Apparatus. The target of this system is a eukaryotic cell. Naturally it is used to inject virulence effector proteins. Last year, we showed that T3SS works in ''E. coli'' by injecting GFP into RK13 cells.
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See [[Team:HokkaidoU_Japan/Project/T3SS|here]] for details about T3SS and our achievements on iGEM 2010.
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See [[Team:HokkaidoU_Japan/Project/T3SS|here]] for the details about T3SS and our achievements on iGEM 2010.
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=Plasmid Backbone for protein injection=
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=Injection assay using onion cells=
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[[File:HokkaidoU_Japan_2011_GSK_Backbone_lv.png|thumb|500px|Fig. 1 A backbone under constitutive promoter(pTetr). Has SlrP as a injection signal, GSK tag, Bsa I Cloning Site. Desired protein can be inserted into the cloning site.]]
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We developed a new method of injection assay using onion cells to evaluate the functions, which is a easier than using mammalian cultured cells. However it requires some treatments specific to plant cells.  
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Last year we made injectable GFP as a reporter of injetion assay. This year, our project started with an aim to conduct direct reprogramming as an application of T3SS.  
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However, attaching signal peptide needed for secretion (T3S signal) and each protein would have been a laborious task. So ready-to-inject backbone was proposed. To accomplish this, we designed Bsa I Cloning Site and developed plasmid backbone which can facilitate quick assembly of proteins to inject (Fig. 1).
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See [[Team:HokkaidoU_Japan/Project/Onion|here]] for the details about Injection assay using onion cells.
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See [[Team:HokkaidoU_Japan/Project/Backbone|here]] for details about plasmid backbone and Bsa I cloning site.
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=Plasmid Backbone for protein injection=
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==Bsa I cloning site==
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[[File:HokkaidoU_BsaI_Backbone.png|thumb|500px|Fig. 1 Ready-to-inject backbone. SlrP is an T3SS injection signal. Bsa I Cloning Site is used for inserting various BioBricks directly. A protein fused to T3S signal can be expressed under control of constitutive promoter(pTetR).]]
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Another problem was how to check if desired protein was definitely injected when it doesn't show any visual effect unlike GFP. To solve this problem, we used a distinct property of Glycogen Synthase Kinase 3β (GSK-3β). GSK-3β is phosphorylated only in eucaryotic cells. By using it as a tag and by detecting its phosphorylation, we can evaluate its translocation into the eukaryotic cell.
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Last year we made injectable GFP as a reporter of injection assay. This year, our project started with an aim to conduct direct reprogramming as an application of T3SS. But first we wanted to try injecting various proteins.
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See [[Team:HokkaidoU_Japan/Project/GSK|here]] for details about GSK tag.
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----
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Fusion of signal peptide, required for secretion , to each protein would have been a laborious task. So development of ready-to-inject backbone was anticipated. To accomplish this, we designed Bsa I Cloning Site and developed plasmid backbone which can facilitate quick assembly of to be injected proteins (Fig. 1).
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We combining these futures in a backbone and constructed ready-to-inject bakcbone. SlrP is an injection signal, without it the protein cannot be secreted. GSK is a tag, by detecting phosphorylation of it you can distinguish whether it has been it eukaryotic cell. For us it is an evidence of successful injection. Bsa I Cloning Site is used for inserting various BioBrick while retaining the whole constructs BioBrick properties. The whole proteins is under control of pTetr constitutive Promoter.
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We used ready-to-inject backbone to inject five proteins representing different structures and functions to see which get injected. This was are shot to try and characterise T3SS a little bit further.
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See [[Team:HokkaidoU_Japan/Project/Backbone|here]] for the details about plasmid backbone and Bsa I cloning site.
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See [[HokkaidoU Japan/Project/GSK#Investigation of T3SS-injectable proteins|here]] for details.
 
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==GSK Tag system==
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[[File:HokkaidoU_Japan_2011_GSK_Backbone_lv.png|thumb|500px|Fig. 2 GSK is a tag for detecting phosphorylation of it in eukaryotic cells.]]
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Another problem is how to check if a protein was successfully injected. GFP is easy, but what about the ones that cannot be visualised? To solve this problem, we used a distinct property of Glycogen Synthase Kinase 3β (GSK-3β). It is phosphorylated only in eucaryotic cells. By fusing a small part of GSK-3β as a tag and by detecting its state of phosphorylation, we can know if and what amount of protein has been injected into the eukaryotic cells (Fig. 2).
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See [[Team:HokkaidoU_Japan/Project/GSK|here]] for the details about GSK tag.
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</div>
{{Team:HokkaidoU_Japan/footer}}
{{Team:HokkaidoU_Japan/footer}}

Latest revision as of 10:48, 15 December 2011

Contents

Abstract

Bacteria living around us evolved ways to effect their surrounding environment. Some bacteria can change its surroundings by injecting whole protein molecules into targeted eukaryotic cells through Type 3 secretion system (T3SS). During iGEM 2010 we showed that E. coli containing a part of Salmonella genome expresses T3SS. We thought this system can be applied to direct reprogramming of somatic cells from eukaryote. This year we tried to make it more convenient. For this purpose we designed a plasmid backbone which can instantly produce ready-to-inject fusion proteins from biobrick parts. Using it, we tried further characterization of this system by injecting various proteins to see if they can be secreted.

What`s T3SS?

T3SS is a system of pathogenic gram-negative bacterium such as Salmonella, Yersinia and EPEC (entero pathogenic E. coli). Using this system bacteria can inject whole protein molecules through a syringe like organelle named T3S Apparatus. The target of this system is a eukaryotic cell. Naturally it is used to inject virulence effector proteins. Last year, we showed that T3SS works in E. coli by injecting GFP into RK13 cells.

See here for the details about T3SS and our achievements on iGEM 2010.

Injection assay using onion cells

We developed a new method of injection assay using onion cells to evaluate the functions, which is a easier than using mammalian cultured cells. However it requires some treatments specific to plant cells.

See here for the details about Injection assay using onion cells.

Plasmid Backbone for protein injection

Bsa I cloning site

Fig. 1 Ready-to-inject backbone. SlrP is an T3SS injection signal. Bsa I Cloning Site is used for inserting various BioBricks directly. A protein fused to T3S signal can be expressed under control of constitutive promoter(pTetR).

Last year we made injectable GFP as a reporter of injection assay. This year, our project started with an aim to conduct direct reprogramming as an application of T3SS. But first we wanted to try injecting various proteins.

Fusion of signal peptide, required for secretion , to each protein would have been a laborious task. So development of ready-to-inject backbone was anticipated. To accomplish this, we designed Bsa I Cloning Site and developed plasmid backbone which can facilitate quick assembly of to be injected proteins (Fig. 1).

See here for the details about plasmid backbone and Bsa I cloning site.


GSK Tag system

Fig. 2 GSK is a tag for detecting phosphorylation of it in eukaryotic cells.

Another problem is how to check if a protein was successfully injected. GFP is easy, but what about the ones that cannot be visualised? To solve this problem, we used a distinct property of Glycogen Synthase Kinase 3β (GSK-3β). It is phosphorylated only in eucaryotic cells. By fusing a small part of GSK-3β as a tag and by detecting its state of phosphorylation, we can know if and what amount of protein has been injected into the eukaryotic cells (Fig. 2).

See here for the details about GSK tag.

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