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...

  1. How to Monitor Cdc28 and Fus3 levels in yeast: File:Fus3cdc28.doc
  2. How to Get Linear Expression of Phospholocator: File:Linearexpr.doc

Protocols:


Plan of Attack

Current Thinking

Schedule for June 14 - June 23

Schedule for June 24 - June 30

Schedule for July 1 - July 8

Schedule for July 9 - July 16

Schedule for July 17 - July 26

Schedule for July 27 - August 2

Schedule for August 6 - August 14

References

Swi5 NLS

  1. 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 | PubMed ID:1652372 | HubMed [Swi5]


Fus3 Docking Site

  1. 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 | PubMed ID:11134045 | HubMed [Fus3Dock]

Swapping Docking Sites

  1. 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 | PubMed ID:10922375 | HubMed [Swap]

Oriented Peptide Library Screens

  1. 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 | PubMed ID:10945990 | HubMed [YaffeCantley]

Chemical Inhibitors of Kinase function

  1. 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 | PubMed ID:11014197 | HubMed [Shokat]

Gel Shift Paper

  1. 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. PubMed ID:9539416 | HubMed [Shift]

Cool Modular Protein Engineering Examples

  1. Array. . PubMed ID:16602891 | HubMed [Overview]
  2. 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 | PubMed ID:14512628 | HubMed [Allostery]
  3. 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 | PubMed ID:12511654 | HubMed [Scaffold]

All Medline abstracts: PubMed | HubMed