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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!

GSK tag

Glycogen Synthase Kinase 3β is known to be phosphorylated by several enzymes in eukaryotic cell. We used first 13 amino acid as a tag (GSK tag)[1]. Ninth amino acid, serine is phosphorylated in eukaryotic cell(Fig). This phosphorylation state could be detected by using phsopho-specific antibodies which bind to only phosphorylated GSK tag. This way it is possible to distinguish whether GSK tag has been it eukaryotic cell. So you can see proteins which were injected into cell and which were not. This was a vital ingredient in our experiments.

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.

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 opted to insert it between SlrP secretion tag and the protein we wanted to inject.

Using non-phosphospecific antibodies it is possible to check the total amount of expressed protein with the tag. Comparing it with the injected protein you can determine the efficiency of the injection.

By comparing the mass of the protein with GSK tag it is also possible to see if it had been modified in eucaryotic cell. It can be used alongside of TEV site and provide proof for successful TEV protease activity. An experiment we would like to try in the future.

Investigation of T3SS-injectable proteins

Here we will discuss about the structure of proteins which could be injected or not. We tried five different proteins: mnt repressor, RFP, GFP, Cre DNA recombinase, (CCR5) transmembrane, LacI and Luciferase. All were chosen from biobrick distribution. As these parts are widely used in iGEM studying them would have a bigger impact compared to exotic ones.

Our main concern was not with the size but the stability of proteins. Previous research show that proteins like Zinc-Fingers are very stable and couldn't be injected. Hight stability prevents unfolding by T3SS chaperons.

We showed that GFP can be injected into eucaryotic cells by confocal laser microscope imaging. Thus it can serve as a control. Next is RFG, a fluorescent protein but with different structure from GFP.

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. [http://www.ncbi.nlm.nih.gov/pubmed/16988240 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. [http://www.ncbi.nlm.nih.gov/pubmed/21498649 PubMed]