HokkaidoU Japan

iGEM 2011 Team of Hokkaido University

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.

Function of 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.

Injection assay using onion cells

We developed a new method of injection assay using onion cells, which is a easier way to evaluate the functions of T3SS than using mammalian cultured cells. But it requires some different treatments that are specific to plant cells.

See here for details about Injection assay using onion cells.

Plasmid Backbone for protein injection

Bsa I cloning site

Fig. 1 Ready-to-inject backbone. An fused protein to T3S signal can be expressed 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.

Since, fusion of signal peptide required for secretion (T3S signal) to each protein would have been a laborious task, development of ready-to-inject backbone has been anticipated. To accomplish this, we designed Bsa I Cloning Site and developed plasmid backbone which can facilitate quick assembly of proteins to be injected (Fig. 1).

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

GSK Tag system

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

Another problem is the way to check if desired protein was definitely injected when it is not visible 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 fusing a small part of GSK-3β as a tag and by detecting its phosphorylation, we can evaluate amount of protein injected into the eukaryotic cells (Fig. 2).

See here for details about GSK tag.

SlrP is an injection signal. GSK is a tag for detecting phosphorylation of it in eukaryotic cells. Bsa I Cloning Site is used for inserting various BioBrick directly. The whole proteins is under control of pTetr constitutive Promoter.

We tested ready-to-inject backbone if it can inject five proteins representing different structures and functions to evaluate ability of the protein injector. This was are shot to try and characterize T3SS a little bit further.

See here for details.