Date of Award

6-2009

Document Type

Union College Only

Degree Name

Bachelor of Science

Department

Biology

Language

English

Keywords

cpps, assay, cargo, cells, membrane

Abstract

Cell Penetrating Peptides (CPPs) are short amino acid sequences that are able to traverse the cell membrane and bring with them attached cargo. Because the membrane acts as a protective barrier, CPPs are potential tools for drug delivery. The Kehlbeck lab began synthesis of a novel CPP, while we, in the Danowski lab designed cellular uptake assays in order to compare them to known CPPs. The ideal assay will allow us to compare uptake efficiency and follow the cargo, from endocytic uptake, to release in the cytoplasm. In this study I used rhodamine to determine optimal fluorophore (cargo) concentration for visualization in cells. Results with unconjugated rhodamine showed that optimal conditions are overnight incubation at a concentration of 0.02 uM. We then did a pilot study with Snap-Tag (New England BioLabs) as a first step in reaching a more biological assay. Cells were transfected with a plasmid encoding the Cytochrome c oxidase enzyme. The transfected cells were given a fluorescent substrate molecule that becomes covalently attached to Cytochrome c oxidase and trapped in the cytoplasm. We will modify this assay, by attaching a membrane-impermeant fluorescent substrate molecule to our CPPs. This approach is promising but requires further work to achieve higher transfection rates.

Download
Off-Campus Download

Share

COinS