Team:HokkaidoU Japan/Project

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=Abstract=
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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 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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== &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Intro ==
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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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&nbsp;&nbsp;&nbsp;Bacteria living around us evolved ways to effect their surrounding environment. This is done by using secretion systems. There are six known types of secretion systems: one through six. These are called Type One Secretion System (T1SS), T2SS, T3SS and so on. Some system only secrete designated proteins outside others can translocate proteins directly to other cells. Secretion systems able to inject proteins into other cell caught many researchers imagination.  
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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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&nbsp;&nbsp;&nbsp;Ours was no exception. We chose to use T3SS for our experiments. During iGEM 2010 we found that E.Coli with a part of Salmonella genome library expresses T3SS. This presented opportunity to work with the amazing machinery without involving pathogenic bacteria.  
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=Injection assay using onion cells=
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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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&nbsp;&nbsp;&nbsp;We repeated previous competitions experiment, one more time showing that GFP can be really injected into a target cell with it. And then continued to characterise it. An further develop data sheet of T3SS possibilities.
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See [[Team:HokkaidoU_Japan/Project/Onion|here]] for the details about Injection assay using onion cells.
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== &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Type Three Secretion System Data Sheet ==
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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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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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Still to come.
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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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== &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Plasmid Backbone for protein injection ==
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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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&nbsp;&nbsp;&nbsp;We developed plasmid backbone which can attach tags needed for secretion and various other functions to a chosen protein biobrick. This can be used for big scale screening of various protein domains for their inject-ability.
 
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More info still to come.
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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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== &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Structure of Injectable Proteins ==
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See [[Team:HokkaidoU_Japan/Project/GSK|here]] for the details about GSK tag.
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Here we will discus the structure of proteins which are injected. Still to come.
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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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