Lissa1: Project Page
What is the Point of this Project?
Debugging!
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...
- How to Monitor Cdc28 and Fus3 levels in yeast: File:Fus3cdc28.doc
- How to Get Linear Expression of Phospholocator: File:Linearexpr.doc
Protocols:
- 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
Schedule for June 14 - June 23
Schedule for June 24 - June 30
Schedule for July 17 - July 26
Schedule for July 27 - August 2
Schedule for August 6 - August 14
References
Swi5 NLS
- Moll T, Tebb G, Surana U, Robitsch H, and Nasmyth K. The role of phosphorylation and the CDC28 protein kinase in cell cycle-regulated nuclear import of the S. cerevisiae transcription factor SWI5. Cell. 1991 Aug 23;66(4):743-58. DOI:10.1016/0092-8674(91)90118-i |
Fus3 Docking Site
- Bardwell AJ, Flatauer LJ, Matsukuma K, Thorner J, and Bardwell L. A conserved docking site in MEKs mediates high-affinity binding to MAP kinases and cooperates with a scaffold protein to enhance signal transmission. J Biol Chem. 2001 Mar 30;276(13):10374-86. DOI:10.1074/jbc.M010271200 |
Swapping Docking Sites
- Smith JA, Poteet-Smith CE, Lannigan DA, Freed TA, Zoltoski AJ, and Sturgill TW. Creation of a stress-activated p90 ribosomal S6 kinase. The carboxyl-terminal tail of the MAPK-activated protein kinases dictates the signal transduction pathway in which they function. J Biol Chem. 2000 Oct 13;275(41):31588-93. DOI:10.1074/jbc.M005892200 |
Oriented Peptide Library Screens
- Obata T, Yaffe MB, Leparc GG, Piro ET, Maegawa H, Kashiwagi A, Kikkawa R, and Cantley LC. Peptide and protein library screening defines optimal substrate motifs for AKT/PKB. J Biol Chem. 2000 Nov 17;275(46):36108-15. DOI:10.1074/jbc.M005497200 |
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. A chemical switch for inhibitor-sensitive alleles of any protein kinase. Nature. 2000 Sep 21;407(6802):395-401. DOI:10.1038/35030148 |
Gel Shift Paper
- Li E, Meldrum E, Stratton HF, and Stone DE. Substitutions in the pheromone-responsive Gbeta protein of Saccharomyces cerevisiae confer a defect in recovery from pheromone treatment. Genetics. 1998 Mar;148(3):947-61. DOI:10.1093/genetics/148.3.947 |
Cool Modular Protein Engineering Examples
- Dueber JE, Yeh BJ, Chak K, and Lim WA. Reprogramming control of an allosteric signaling switch through modular recombination. Science. 2003 Sep 26;301(5641):1904-8. DOI:10.1126/science.1085945 |
- Park SH, Zarrinpar A, and Lim WA. Rewiring MAP kinase pathways using alternative scaffold assembly mechanisms. Science. 2003 Feb 14;299(5609):1061-4. DOI:10.1126/science.1076979 |
