Date Approved

2004

Degree Type

Open Access Senior Honors Thesis

Department

Chemistry

Abstract

This project focuses on the development of an undergraduate-level laboratory procedure involving EMU's new automated DNA sequencer. The gene used for this procedure is CYP2B4 from the rabbit genome, which codes for Cytochrome P450-2B4 (CVP450). Cytochrome P450s are an important class of proteins found in many species throughout the animal kingdom, including humans. The wild-type CYP2B4 will be sequenced, and then mutated to replace threonine-302 with alanine. Threonine-302 is suspected to playa key role in P450 function. Thus far, procedures have been developed for site-directed mutagenesis to convert threonine-302 to an alanine residue, transformation of bacteria with the target gene, isolation of the plasmid from the transformed bacteria, harvesting the bacteria to express the gene, and preparation of the protein for spectrophotometric analysis. While development of a viable DNA sequencing protocol has met with limited success, the protocol used for the site-directed mutagenesis of CVP2B4 has brought promising results.

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