Secrets of the rainforest

Tisha’s Story

GFP – Where Does It Come From? Jellyfish: Aequorea victoria Used for: Means of visual communication Predation Mating Symbiosis Warning signal

GFP Structure – The Beta Barrel 238 amino acids Cylindrical fold Very stable structure that is resistant to denaturing Alpha helices are red and beta pleated sheets are green.

Why a Black Light? The flurophore is embedded in the beta barrel structure In the Organism An influx of Ca+2 causes the first protein, aequorin, to become excited and transfer the energy to GFP GFP loses the energy by emitting a photon of green light

What’s a Reporter Molecule? A reporter molecule is one protein (like GFP) linked to the protein you are interested in studying. You can follow what your protein is doing by following the reporter molecule (GFP).

GFP As A Biological Tracer

Our experiment

Step 1 – Bacterial Culturing

Today – Plate culture Used to isolate individual colonies

How do you streak a plate?

Tomorrow – broth culture Used to grow many of one particular colony (same genetic makeup)

Step 2 – Lyse Bacterial Cells (to extract protein)

How will we lyse the cells? Centrifuge Lysosyme Freezer

Step 3 – GFP Purification via Hydrophobic Interaction Chromatography

How does the HIC column work?

HIC Step 1 Add bacterial lysate to column matrix in high salt buffer Hydrophobic proteins interact with column Salt ions interact with the less hydrophobic proteins and H2O Hydrophobic bead N H + - O S N H + - O S

HIC Step 2 Wash less hydrophobic from column with low salt buffer bead Wash less hydrophobic from column with low salt buffer Less hydrophobic E. coli proteins fall from column GFP remains bound to the column - + - + N H + - O S - +

HIC Step 3 Elute GFP from column by adding a no-salt buffer GFP Hydrophobic bead Elute GFP from column by adding a no-salt buffer GFP Released from column matrix Flows through the column - + - + - + - + - +

Helpful HIC Tips Add a small piece of paper to collection tube where column seats to insure column flow Rest pipet tip on side of column to avoid column bed disturbance when adding solutions Drain until the meniscus is just above the matrix for best separation