HokkaidoU Japan

iGEM 2011 Team of Hokkaido University

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• Abstract
• What`s T3SS
  Detailed information about T3SS and summary of our achievements on iGEM 2010
• Injection assay using onion cells
  Experiments using plant cells are easier to perform than with mammalian ones
• Ready-to-inject backbone and Bsa I cloning site
  Ready-to-inject backbone and Bsa I cloning site enables easy fusion of T3S signal and protein
• GSK tag system
  A neat injection assay using GSK tag, which can specifically detect successfully injected proteins
• Bsa I cloning site, RFC submission
  Detailed documentation of costructing a BioBrick cloning site a BioBrick!

Abstract

Bacteria living around us evolved ways to effect their surrounding environment. Some bacteria can change its surrounding environment by injecting whole protein molecules into targeted eukaryotic cells throug 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.

Introduction to 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 details about T3SS and our achievements on iGEM 2010.

Plasmid Backbone for protein injection

Bsa I cloning site

Fig. 1 Ready-to-inject backbone. An inserted protein with T3S signal is under control of constitutive promoter(pTetR).

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.

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).

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

GSK Tag system

Fig. 2 GST tag is added between T3S signal and Bsa I cloning site.

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). It is phosphorylated only in eucaryotic cells. By using a small part of GSK-3β as a tag and by detecting its phosphorylation, we can evaluate its translocation into the eukaryotic cell (Fig. 2).

See here for details about GSK tag.

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.

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.

See here for details.