Document Type

Honors Capstone Project

Date of Submission

Spring 5-1-2008

Capstone Advisor

Mark S Braiman

Honors Reader

Michael S Cosgrove

Capstone Major


Capstone College

Arts and Science

Audio/Visual Component


Capstone Prize Winner


Won Capstone Funding


Honors Categories

Sciences and Engineering

Subject Categories

Biochemistry | Biochemistry, Biophysics, and Structural Biology


Much of what is currently known about GPCR structure was based on X-ray crystallographic measurements of rhodopsin. Furthermore, due to its inexpensiveness and the availability of simple methods to purify it in large quantities from cow eyes, bovine rhodopsin has been successfully used in obtaining diffraction-quality three-dimensional crystals. In our experiment, the bovine rhodopsin protein was cloned into the commercially available E. coli expression plasmid, pBAD TOPO®, using forward and reverse PCR primers designed in our lab. Plasmid DNA from these cells was purified in order to analyze the direction of the inserted gene. The purified plasmid DNA was digested with NcoI, BamHI, and HindIII to assess the orientation of the bovine rhodopsin gene. In addition, the gene was sequenced. Once the correct orientation of the inserted gene was ascertained, E.coli cells strain UT5600 were transformed with the purified plasmid DNA. A 350-ml culture of cells from the transformation reaction was grown. L-arabinose and retinal were added to the cells to induce expression of the rhodopsin protein. In analyzing the cells, an orange-brown color was observed instead of the anticipated red color, which we hypothesize to be the binding of all-trans retinal to the opsin apoprotein in rhodopsin.

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This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

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