Section 5 Group 21
Monika Malek: 25%
Megan O’Connor: 25%
Lexy Petrick: 25%
Sophie Sampson: 25%
Experimental Plan
Abstract:
We will explore the relationship between the proteins F58E10.3 and LIN-66, previously identified as a prey and bait, respectively, in a two-hybrid screen. We explore this relationship through a GST pull-down assay, analyzed by gel electrophoresis. If the proteins successfully bind, we expect to see the eluted and bound together GST/bait/prey complex on gel electrophoresis, followed by a final western blot assay. Confirmation of these two proteins interacting is important for controlling developmental timing in C. elegans.

Material and Methods:
MagneGST™ Pull-Down System (purchased from the Promega Catalog), which includes the below listed components: * Methionine * RQ1 RNase-Free DNase * Nuclease-Free Water * MagneGST™ Cell Lysis Reagent * MagneGST™ Glutathione Particles * MagneGST™ Binding/Wash Buffer * TnT® T7 Quick Master Mix
These components can be purchased from the Promega catalog as well; * E. coli KC8 strain (hsdR, leuB600, trpC9830, pyrF::Tn5, hisB463,lacΔX74, strA, galU,K, lac) * Agarose gel * Gel electrophoresis ladder * 2X SDS gel loading buffer * Western blot reagents
The following components will be synthesized in our lab: * Appropriate GST vector containing coding sequences for lin-66 * F58E10.3 prey protein obtained from C. elegans DNA

Experimental Plan:
To show the protein F58E10.3 binds to LIN-66, we will test their interaction in a GST pull-down assay (Einarsen, 2001; Promega Corporation, 2007). To do so we will have to; * Clone a lin-66 bait protein into a vector containing a GST (glutathione-S-transferase) tag and the appropriate elements for growth plus protein expression in E. coli. * Establish the GST bait fusion protein is being