Lissa1: Project Page
What is the Point of this Project?
The goal of this project is to characterize the inner workings of the phospholocator , designed and built by Samantha Sutton. Although we know how the phospholocator device works, we don't have a quantitative description of how the parts interact.
Since the effectiveness (gain) of the phospholocator (PPL) device depends on the kinase parts being active, or phosphorylated, I will spend this summer finding out:
- how much phosphorylated kinase is present in a cell
- the relationship between phosphorylated kinase and phosphorylated PPL
- how changing the concentration of active kinase and PPL in the cell effects the gain of the system
- how to use this knowledge (which will be collected and posted on the [Registry of Standard Biological Parts]) to increase the gain of the PPL
- How to Monitor Cdc28 and Fus3 levels in yeast: Image:Fus3cdc28.doc
- How to Get Linear Expression of Phospholocator: Image:Linearexpr.doc
- Western Blot
- Sumo gels
- Overnight Culture
- Plasmid Digests
- Pouring Plates
- Protein Extracts
- Yeast Transformations
- EZ Yeast Transformations
- Miniprep - see QIAGEN manual
- Small Agarose Gels
- Setting up PCR reactions
- Native Extraction
Plan of Attack
- Moll T, Tebb G, Surana U, Robitsch H, and Nasmyth K. . pmid:1652372.
Fus3 Docking Site
Swapping Docking Sites
Oriented Peptide Library Screens
- Obata T, Yaffe MB, Leparc GG, Piro ET, Maegawa H, Kashiwagi A, Kikkawa R, and Cantley LC. . pmid:10945990.
Chemical Inhibitors of Kinase function
- Bishop AC, Ubersax JA, Petsch DT, Matheos DP, Gray NS, Blethrow J, Shimizu E, Tsien JZ, Schultz PG, Rose MD, Wood JL, Morgan DO, and Shokat KM. . pmid:11014197.
Gel Shift Paper
- Li E, Meldrum E, Stratton HF, and Stone DE. . pmid:9539416.
Cool Modular Protein Engineering Examples