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100 nm

3 um

1 um E.coli

= 2 nm

80 nm

Protein delivery into eukaryotic cells by type III secretion machines Jorge E. Galan and Hans Wolf-Watz, 2006, Nature 444: 567-573

About T3SS

Bacterial cytosol

Target cell cytosol

Target cell membrane

Inner membrane

Outer membrane

pSB1C3

E. coli K-12 [SPI-2/T3Signal-GFP/RFP]

pBAC SPI2

secretion apparatus

target cell

pSB1C3

Promoter RBS T3S signal GFP Double

terminator RFP

reporter RFP

reporter T3S signal GFP

Fused protein

GFP

GFP

Gene Construct

Result

Merge GFP

RFP phase-contrast

Objectives for iGEM 2011

I. Making injection assay easier by using onion cells sheets

II. Screening what proteins can be injected by T3SS

III. Seeking a method for verifying the injection of proteins

Animal cells Onion cells

Difficult to prepare

Weak against bacteria

Easy to prepare

Tolerant against bacteria

1 2 3

4 5

6 7

Result

A B C

Objectives for iGEM 2011

I. Making injection assay easier by using onion cells sheets

II. Screening what proteins can be injected by T3SS

III. Seeking a method for verifying the injection of proteins

Wish to clone various proteins into…

HERE

S N P SlrP sig.

TetR

RBS E N X

Double Terminator

Biofusion standerd takes…

BioBrick

Biofusion Prefix primer

Biofusion Suffix primer

Too much cost and time!

E N X S N P

What should we do?

Four requirements for cloning system

I. Inserting various kinds of BioBrick parts into another Biobrick using a SAME primer set for PCR.

II. Making it sure that the part is to be inserted in the correct direction.

III. Producing a fusion protein.

IV. Resulting construct to retain BioBrick properties.

Objectives for iGEM 2011

I. Making injection assay easier by using onion cells sheets

II. Screening what proteins can be injected by T3SS

III. Seeking a method for verifying the injection of proteins

Glycogen Synthase Kinase 3β

GSK tag and location

Bacterial cytosol

Eukaryotic cytosol

Phosphorylated NO YES

unmodified YES NO

Location

GSK Tag state

Julie Torruellas Garcia, Franco Ferracci, Michael W. Jackson,1 Sabrina S. Joseph, Isabelle Pattis, Lisa R. W. Plano, Wolfgang

Fischer, and Gregory V. Plano. 2006. Measurement of Effector Protein Injection by Type III and Type IV Secretion Systems by

Using a 13-Residue Phosphorylatable Glycogen Synthase Kinase Tag. Infect Immun.Vol.74:5645-57.

GSK tag and antibodies

Anti phosphorylated

GSK Anti GSK

Phosphorylated Recognizes Recognizes

unmodified - Recognizes

Atibodies

GSK Tag state

Julie Torruellas Garcia, Franco Ferracci, Michael W. Jackson,1 Sabrina S. Joseph, Isabelle Pattis, Lisa R. W. Plano, Wolfgang

Fischer, and Gregory V. Plano. 2006. Measurement of Effector Protein Injection by Type III and Type IV Secretion Systems by

Using a 13-Residue Phosphorylatable Glycogen Synthase Kinase Tag. Infect Immun.Vol.74:5645-57.

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