Team:HokkaidoU Japan

From 2010.igem.org

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[[Team:HokkaidoU_Japan|English]]
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[[Team:HokkaidoU_Japan/home_jp|日本語]]
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=[[Team:HokkaidoU_Japan/Projects|Projects]]=
=[[Team:HokkaidoU_Japan/Projects|Projects]]=
<div id="projects" class="homepics">[[Team:HokkaidoU_Japan/Projects| ]]</div>
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==Dr. ''E.coli'' : The smallest protein injector in the world==
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==[[Team:HokkaidoU_Japan/Projects|Dr. ''E. coli'' : The smallest protein injector in the world]]==
&nbsp;&nbsp;&nbsp;Our main project is on Type III Secretion Apparatus which is one of the most amazing biological devices. This apparatus which looks like a syringe can pass a whole protein molecule from a bacterial cell to a target eukaryotic cell. However, this apparatus is an organelle of pathogenic gram-negative bacterium such as ''Salmonella'' and ''Yersinia''. So we are aiming at making this device safely available using ''E. coli''.
&nbsp;&nbsp;&nbsp;Our main project is on Type III Secretion Apparatus which is one of the most amazing biological devices. This apparatus which looks like a syringe can pass a whole protein molecule from a bacterial cell to a target eukaryotic cell. However, this apparatus is an organelle of pathogenic gram-negative bacterium such as ''Salmonella'' and ''Yersinia''. So we are aiming at making this device safely available using ''E. coli''.
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==[[Team:HokkaidoU_Japan/Projects/PCR|PCR Based Assembly Protocol]]==
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==How does it function?==
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&nbsp;&nbsp;&nbsp;Sometimes standard protocol fails to produce plasmid or BioBrick is toxic to host cell and can't be amplified. In moments like these PCR is handy. We think we might slightly improved it. You no longer have to guess if restriction was successful.
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&nbsp;&nbsp;&nbsp;When the needle tip attaches to the host cell membrane a translocator complex that is also secreted by the T3SS is assembled on the host cell membrane and mediates the passage of the effector proteins through the target cell membrane. On the other hand an effector protein, which have a unique T3SS secretion signal domain on its N-terminal, is recognized by the specific chaperone and form an effector-chaperone complex. The secretion machinery, including a T3-secretion-associated ATPase, recognizes the complex. Then, the ATPase stripes the chaperone from the complex, which remains within the bacterial cell, and mediates the unfolding and threading of the effector protein through the central channel of the needle complex. Finally, the translocated effectors re-fold within the host cell to carry out their function. (Fig.2)
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==Motivation==
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&nbsp;&nbsp;&nbsp;It is valuable to develop a system that can modulate cells’ behavior impersistently by injecting a desired “protein” directly into cells using a non-pathogenic strain. This system can be applied for many ways. For example,
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– Inject p53 to kill cancer cells selectively
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– Inject Yamanaka factors to induce iPS cells
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And fortunately It was reported in 2007 that T3SS encoded in Salmonella Pathogenesity Island 2(SPI2) is functional in vitro on the E.coli(K-12) strain.(9) However, the T3SS encoded in SPI-2 naturally function inside of the phagosome of the target cell.(8) So, there was no report about whether the SPI-2 T3SS, that is cloned on E.coli(K-12), can inject a heterologous protein from outside of the target cell or not.
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That is why we have decided to put this challenging project into practice.
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==Objectives of iGEM 2010==
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==Construction of a PCR based protocol==
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あれやこれや
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&nbsp;&nbsp;&nbsp;The Hokkaido University's igem 2010 team, HokkaidoU_Japan is Japanese 8th the youngest team, consisting of one instructer and 7 undergrad students from facalty of Sciences, Medicine and Agriculture. This is the first year for us to participate in iGEM competition.
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&nbsp;&nbsp;&nbsp;The Hokkaido University's igem 2010 team, HokkaidoU_Japan is Japans 8th Our has one instructer and 7 undergrad students.They come from faculty of Sciences, Medicine and Agriculture. This is the first year we are participating in iGEM.
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=[[Team:HokkaidoU_Japan/Notebook|Notebook]]=
=[[Team:HokkaidoU_Japan/Notebook|Notebook]]=
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<div id="notebook" class="homepics">[[Team:HokkaidoU_Japan/| ]]</div>
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&nbsp;&nbsp;&nbsp;The [[Team:HokkaidoU_Japan/Notebook|Notebook]] is a record of repeat of failures.  
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&nbsp;&nbsp;&nbsp;
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The record of our triumphs and failures. Well big part of it is failures. But we did it.
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<div id="protocols" class="homepics">[[Team:HokkaidoU_Japan/Protocols| ]]</div>
<div id="protocols" class="homepics">[[Team:HokkaidoU_Japan/Protocols| ]]</div>
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General protocols we used.
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=[[Team:HokkaidoU_Japan/Parts|Parts]]=
=[[Team:HokkaidoU_Japan/Parts|Parts]]=
<div id="parts" class="homepics">[[Team:HokkaidoU_Japan/Parts| ]]</div>
<div id="parts" class="homepics">[[Team:HokkaidoU_Japan/Parts| ]]</div>
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&nbsp;&nbsp;&nbsp;We registered two parts associated with main project. One is a signal sequence that can be used to secrete tagged proteins through Type III Secretion Apparatus. The other is whole contruct that checks whether the Type III secretion system works correctly.
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&nbsp;&nbsp;&nbsp;We registered two parts associated with main project. One is a signal sequence that can be used to secrete tagged proteins through Type III Secretion Apparatus. The other is whole reporter construct to checks whether the Type III secretion system works correctly.
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&nbsp;&nbsp;&nbsp;Also, we registerd one primer set that is useful for the PCR based protocol.
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&nbsp;&nbsp;&nbsp;Also, we registerd one primer set which is useful for the PCR based protocol.
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=[[Team:HokkaidoU_Japan/Safety|Safety]]=
=[[Team:HokkaidoU_Japan/Safety|Safety]]=
<div id="safety" class="homepics">[[Team:HokkaidoU_Japan/Safety| ]]</div>
<div id="safety" class="homepics">[[Team:HokkaidoU_Japan/Safety| ]]</div>
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&nbsp;&nbsp;&nbsp;なんとやら
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&nbsp;&nbsp;&nbsp;Addressing bio safety concerns
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<br><br><br><br>
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=Acknowledgements=
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== Laboratory and Personnel ==
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* Ken-ichi Yamazaki
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** Associate Professor/Environmental Molecular Biology/Graduate School of Environmental Earth Science/Hokkaido University
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*Hideaki Higashi
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** Associate Professor /Division of Molecular Oncology/ Institute for Genetic Medicine/ Hokkaido University
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* Hisatoshi Shida
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** Professor/Molecular Biology/Institute for Genetic Medicine/ Hokkaido University
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* Takashi Oohashi
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** Associate Professor /Division of Molecular Oncology/ Institute for Genetic Medicine/ Hokkaido University
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* Members of Yamazaki Lab
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*Masaaki　Morikawa
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** Professor/Environmental Molecular Biology/Graduate School of Environmental Earth Science/Hokkaido University
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* Hidetoshi Okuyama
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** Associate Professor/Environmental Molecular Biology/Graduate School of Environmental Earth Science/Hokkaido University
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Thank you for letting us use some of the resources or equipment.
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==Academic Institutions==
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* Salmoneｌｌa Genetic Stock Center/University of Calgary
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**Provided E.coli with BAC vector library coding for Type 3 Secretion Apparatus and SlrP signal
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* Graduate School of Environmental Earth Science
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* Department of biological science/School of Sciense
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* Institute for Genetic Medicine
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==Companies==
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* Amino Up Chemical
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* ECONIXE
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* Cosmo Bio
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* Mendel　Workshop
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==Person==
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* Tadasu Emoto
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**Manager of ECONIXE

Latest revision as of 10:51, 21 April 2011

English / 日本語

Projects

Dr. E. coli : The smallest protein injector in the world

   Our main project is on Type III Secretion Apparatus which is one of the most amazing biological devices. This apparatus which looks like a syringe can pass a whole protein molecule from a bacterial cell to a target eukaryotic cell. However, this apparatus is an organelle of pathogenic gram-negative bacterium such as Salmonella and Yersinia. So we are aiming at making this device safely available using E. coli.

PCR Based Assembly Protocol

   Sometimes standard protocol fails to produce plasmid or BioBrick is toxic to host cell and can't be amplified. In moments like these PCR is handy. We think we might slightly improved it. You no longer have to guess if restriction was successful.

Team

   The Hokkaido University's igem 2010 team, HokkaidoU_Japan is Japans 8th Our has one instructer and 7 undergrad students.They come from faculty of Sciences, Medicine and Agriculture. This is the first year we are participating in iGEM.

Notebook

    The record of our triumphs and failures. Well big part of it is failures. But we did it.

Protocols

    General protocols we used.

Parts

   We registered two parts associated with main project. One is a signal sequence that can be used to secrete tagged proteins through Type III Secretion Apparatus. The other is whole reporter construct to checks whether the Type III secretion system works correctly.

   Also, we registerd one primer set which is useful for the PCR based protocol.

Safety

   Addressing bio safety concerns





Acknowledgements

Laboratory and Personnel

  • Ken-ichi Yamazaki
    • Associate Professor/Environmental Molecular Biology/Graduate School of Environmental Earth Science/Hokkaido University
  • Hideaki Higashi
    • Associate Professor /Division of Molecular Oncology/ Institute for Genetic Medicine/ Hokkaido University
  • Hisatoshi Shida
    • Professor/Molecular Biology/Institute for Genetic Medicine/ Hokkaido University
  • Takashi Oohashi
    • Associate Professor /Division of Molecular Oncology/ Institute for Genetic Medicine/ Hokkaido University
  • Members of Yamazaki Lab
  • Masaaki　Morikawa
    • Professor/Environmental Molecular Biology/Graduate School of Environmental Earth Science/Hokkaido University
  • Hidetoshi Okuyama
    • Associate Professor/Environmental Molecular Biology/Graduate School of Environmental Earth Science/Hokkaido University

Thank you for letting us use some of the resources or equipment.

Academic Institutions

  • Salmoneｌｌa Genetic Stock Center/University of Calgary
    • Provided E.coli with BAC vector library coding for Type 3 Secretion Apparatus and SlrP signal
  • Graduate School of Environmental Earth Science
  • Department of biological science/School of Sciense
  • Institute for Genetic Medicine

Companies

  • Amino Up Chemical
  • ECONIXE
  • Cosmo Bio
  • Mendel　Workshop

Person

  • Tadasu Emoto
    • Manager of ECONIXE





Retrieved from "http://2010.igem.org/Team:HokkaidoU_Japan"