Lissa1: Project Page
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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: Image:Fus3cdc28.doc
- How to Get Linear Expression of Phospholocator: Image: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
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 pmid:11134045.
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 pmid:10922375.
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 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. A chemical switch for inhibitor-sensitive alleles of any protein kinase. Nature 2000 Sep 21; 407(6802) 395-401. doi:10.1038/35030148 pmid:11014197.
Gel Shift Paper
Cool Modular Protein Engineering Examples
- Bhattacharyya RP, Reményi A, Yeh BJ, and Lim WA. Domains, Motifs, and Scaffolds: The Role of Modular Interactions in the Evolution and Wiring of Cell Signaling Circuits. Annu Rev Biochem 2006 Mar 17. doi:10.1146/annurev.biochem.75.103004.142710 pmid:16602891.
- 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 pmid:14512628.
- 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 pmid:12511654.
