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Team:HokkaidoU Japan/Project/GSK
From 2011.igem.org
HokkaidoU Japan
iGEM 2011 Team of Hokkaido University
Contents |
GSK tag
GSK tag was constructed by Julie Torruellas Garcia et al. It is phosphorylated only in eucaryotic cells. So, we can figure out if protein was injected in eucaryotic cells by detecting phosphorylated GSK tag. GSK tag is derived from first 13aa of GSK-3β. Because of it's small size, the interference in tagged protein should be at minimum. We removed present Spe I site in the sequence by silent mutation.
It was reported that GSK tag can be added to N terminus, C terminus and anywhere in between of the protein. T3S signal should be on N terminus, we inserted GSK tag between T3S signal and Bsa I cloning site.
Investigation of T3SS-injectable proteins
Here we will discuss the structure of proteins which are injected and which are not. We tried eight different proteins: mnt repressor, Gal4, RFP, GFP, Cre DNA recombinase, (CCR5) transmembrane, LacI and Luciferase. All were chosen from biobrick distribution which shows their significant importance for iGEM.
Our main concern was not with the size the protein but its stability. Previous research show that proteins like Zinc-Finger are were stable and couldn't be injected. Stability prevents unfolding by T3SS chaperons. Our asortment includes Gal4 which is representative of stable proteins.
| Name | Registry | 2011 distribution | length (bp) | total molecular weight (kDa) |
|---|---|---|---|---|
| Discription | ||||
| mnt repressor | [http://partsregistry.org/Part:BBa_C0072 BBa_C0072] | 1-12L | 288 | 42.1 |
| Discription | ||||
| Gal4 DNA binding domain | [http://partsregistry.org/Part:BBa_K105007 BBa_K105007] | 3-9I | 438 | 47.6 |
| Discription | ||||
| RFP | [http://partsregistry.org/Part:BBa_J06504 BBa_J06504] | 1-13F | 714 | 57.7 |
| Discription | ||||
| GFP | [http://partsregistry.org/Part:BBa_E0040 BBa_E0040] | 1-14K | 720 | 57.9 |
| Discription | ||||
| Cre DNA recombinase | [http://partsregistry.org/Part:BBa_J61047 BBa_J61047] | 1-5D | 1037 | 69.6 |
| Discription | ||||
| CCR5 | [http://partsregistry.org/Part:BBa_I712002 BBa_I712002] | 2-3D | 1059 | 70.4 |
| Discription | ||||
| LacI | [http://partsregistry.org/Part:BBa_I732100 BBa_I732100] | 2-10E | 1086 | 71.4 |
| Discription | ||||
| Luciferase | [http://partsregistry.org/Part:BBa_I712019 BBa_I712019] | 1-10H | 1653 | 92.1 |
| Discription | ||||
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]
