School: Shorewood High School
City: Shoreline, WA
Grades taught: 9-10 (Chemistry, Biotechnology)

Principle Investigator: Ken Mackie, Ph.D.
Department: Anesthesiology, School of Medicine
Institution: University of Washington

Project Description
The Mackie Lab studies the functioning of CB-1 receptors—those receptors on neural cells that bind with endocannabinoids.  Although the most commonly known cannabinoid is THC found in marijuana, mammals produce endogenous cannabinoids (endocannabinoids) that are involved in normal brain activity.  The Mackie Lab researches a variety of molecules that interact with the CB-1 receptor and endocannabinoids.

One molecule of interest is fatty acid amide hydrolase (FAAH).  This molecule breaks down anandamide, an endocannabinoid.  There is much speculation about the possible uses of FAAH for pain therapy and the role it might play in the development of drug tolerance. 

The Mackie Lab has developed a polyclonal antibody that binds to FAAH.  By using a fluorescent tag on this antibody, FAAH’s location in the cell can be detected.  Unfortunately, the antibody does not appear to be specific for FAAH as it binds to regions of the neuron not thought to contain FAAH.

My project, then, was to purify the antibody, making it more specific to FAAH.  This was done by subcloning the DNA of two small peptides of FAAH into protein expression vectors, transforming bacterial cells with these vectors, and using these cells to produce a large quantity of the two FAAH peptides.  It was hoped that these small parts of FAAH could be used in affinity columns to purify the polyclonal antibody and extract those antibody molecules that would bind only to FAAH.  Although I was successful in subcloning the DNA, transforming bacterial cells, and producing proteins, I was able to purify only one of the peptides.  That protein is sufficiently pure to use in an affinity column.  After purifying the antibody, it can be applied to cultured neural cells to visualize the location of FAAH.

I hope to use many of the techniques I learned this summer in my biotechnology class at Shorewood High School.  But more importantly, my experience as a researcher has helped me realize the true excitement of finding answers and the frustration of making mistakes and of wrestling with understanding difficult concepts. I hope that all of these insights will make me a more understanding and empathetic teacher.

Acknowledgements
Thanks to everyone in the Mackie Lab for their help and patience.  Special thanks to Jim Wager-Miller and Matt Wallace for all their time and effort in making my research experience so valuable.