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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 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). Phosphorylated serine in the polypeptide could be detected by using phsopho-specific antibodies which bind to only phosphorylated GSK tag. So it is effective 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 alocated the tag between SlrP secretion tag and the protein we to be injected.

Non-phosphospecific antibodies for determination of total amount of expressed fusion protein with the tag we used.

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 propose 8 different proteins: mnt repressor, Gal4 DNA binding domain, RFP, GFP, Cre DNA recombinase, CCR5, LacI and Luciferase. All are 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 is not with the size but the stability of proteins against unfolding by T3SS chaperone. Previous research indicated that proteins containing Zinc-Fingers are very stable and couldn't be injected. Proteins containing such stable structure is thought to resist against unfolding by T3SS chaperon.

So, we have cloned that various kinds of proteins (Table. 1) into the Bsa I cloning site mentioned here, so that the T3SS carrying E. coli can produce a T3S tagged protein.

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

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]