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.

How does it function?

   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)

Motivation

   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, – Inject p53 to kill cancer cells selectively – Inject Yamanaka factors to induce iPS cells 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. That is why we have decided to put this challenging project into practice.

Objectives of iGEM 2010

Construction of a PCR based protocol

あれやこれや

Team

   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.

Notebook

   The Notebook is a record of repeat of failures.

Protocols

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 contruct that checks whether the Type III secretion system works correctly.

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

Safety

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