HokkaidoU Japan

iGEM 2011 Team of Hokkaido University

• Home
• Team
• Project
• Parts
• Protocols
• Notebook
• Safety
• Human Practice

• 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!

GSK tag

Glycogen Synthase Kinase 3β is known to be phosphorylated by several enzymes in eukaryotic cells. We used first 13 amino acid as a tag (GSK tag) of injected fusion protein^[1]. Ninth amino acid, serine, is phosphorylated in eukaryotic cells (Fig. 1). GSK tags phosphorylated state can be specifically detected by using phospho-specific antibodies. So it is effective method to distinguish GSK tag fusion protein existing in eukaryotic cells from uninjected protein remaining E. coli.

GSK tag was constructed by Julie Torruellas Garcia, Gregory V. Plano et al. We removed present Spe I site in the sequence by silent mutation.

Fig. 1
 Translation: M   S   G   R   P   R   T   T   S-p  F   A   E   S
 Original   :ATG AGT GGT CGC CCT CGC ACT ACT  AGT TTC GCT GAA AGT
 rm Spe I   :ATG AGT GGT CGC CCT CGC ACT ACA* AGT TTC GCT GAA AGT 
Phosphorylated Serine is shown as S-p.

GSK tag can be added to N terminus^[1], C terminus^[1], and anywhere in middle^[2], of the protein. We located the tag between SlrP secretion tag and the protein fused to it.

Non-phosphospecific antibodies can used for determination of total amount of expressed fusion protein within the assay.

Construction of GSK tagged T3SS-injectable proteins

Here we show a list of proteins which are to be tested for protein screening using GSK tag. We chose 8 different proteins (Table. 1). All are from iGEM 2011 biobrick distribution kit. As these parts are widely used in iGEM, characterising them would have a bigger impact compared to exotic ones.

Our main concern is not with the size but the stability of proteins against unfolding by T3SS chaperone. Previous research indicate that proteins containing Zinc-Fingers are very stable and couldn't be injected. Proteins containing such stable structure is thought to resist unfolding by T3SS chaperon.

We cloned these proteins into the Bsa I cloning site mentioned here.

We have previously shown that GFP can be injected into eucaryotic cells by observation under confocal laser microscope. Thus we have a positive control.

Table. 1 A list of several recommended proteins to be injected. Total molecular wight of each protein contains T3S signal and GSK tag domain.

However, of the time constraints we done only Pilot test which failed as a experiment for unknown causes so we don`t have a data to show.

To confirm the expression of the GSK tagged SlrP fusion proteins in E. coli bacterial cell lysate must be analyzed by SDS-PAGE and immunoblotting with a GSK-3β(Cell　Signaling Technologies #9332 ) and a phosphospecific GSK-3β(Cell　Signaling Technologies #9336) antibody preparation.

References

• 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. PubMed
• JWensheng Luo and Michael S. Donnenberg. 2011. Interactions and Predicted Host Membrane Topology of the Enteropathogenic Escherichia coli Translocator Protein EspB. J. Bacteriol.Vol.193:2972–80. PubMed