Abstract

Bacteria living around us evolved ways to effect their surrounding environment. This is done by using secretion systems. Type 3 Secretion System can inject whole protein molecules into targeted eukaryotic cells. During iGEM 2010 we showed that E. coli with a part of Salmonella genome library expresses T3SS. We thought this system can be applied to direct reprogramming of somatic cells among many other things. 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 ordinary biobrick part. Using it, we tried to further characterize this system by injecting characteristic 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

Fig. 1 A backbone under constitutive promoter(pTetr). Has SlrP as a injection signal, GSK tag, Bsa I Cloning Site. Desired protein can be inserted into the cloning site.

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.

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). GSK-3β is phosphorylated only in eucaryotic cells. By using it as a tag and by detecting its phosphorylation, we can evaluate its translocation into the eukaryotic cell.

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 [HokkaidoU Japan/Project/GSK#Investigation of T3SS-injectable proteins here] for details.