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...

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:

• 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

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

Error fetching PMID 1652372

Fus3 Docking Site

Error fetching PMID 11134045

Swapping Docking Sites

Error fetching PMID 10922375

Oriented Peptide Library Screens

Error fetching PMID 10945990

Chemical Inhibitors of Kinase function

Error fetching PMID 11014197

Gel Shift Paper

Error fetching PMID 9539416

Cool Modular Protein Engineering Examples

Error fetching PMID 16602891
Error fetching PMID 14512628
Error fetching PMID 12511654