Biochemistry laboratory

Lectures: 6 sessions over 8 weeks, 1 hour / session

Labs: 2 sessions / week, 4 hours / session

The course covers biochemistry laboratory techniques in the context of investigating resistance to the cancer drug Gleevec.

Gleevec is a small molecule drug for chronic myelogenous leukemia (CML) that functions as a potent inhibitor of the protein Bcr-Abl, an aberrant kinase implicated in CML. While many CML patients treated with Gleevec experience remission, a significant population develops resistance to the drug. Point mutations in the gene that encodes the Bcr-Abl protein have been identified in patients with Gleevec-resistant CML.

In this course, students express a Gleevec-resistant mutant of the Bcr-Abl kinase domain and investigate in-vitro inhibition of kinase activity by Gleevec and a second kinase inhibitor, Dasatinib. The mutant Abl domain is expressed in E. coli and then purified and analyzed using nickel affinity chromatography, polyacrylamide gel electrophoresis, UV-Vis spectroscopy, and BSA quantification. Students use a coupled phosphorylation assay to determine the specific activity of their expressed mutant kinase domain and the wild type kinase domain in the absense and presence of Gleevec and Dasatinib. Students use a structure-viewing program to explore the mechanistic basis of Bcr-Abl inhibition and Gleevec-resistance.

Students also use site-directed mutagenesis to create the DNA for another Gleevec-reistance mutant of their choice.

Prior to each laboratory session (except for session 1), you should outline the procedures you will be carrying out and complete any relevant calculations. Prelabs should include a clear outline, but need not repeat detailed procedures that you will refer to in your lab manual (for example, the step by step procedure for a miniprep). Your TA will assign you a grade of 0, 1 or 2 points for each lab session based on your preparation and participation in lab for that day.

The calendar below provides information on the course's lecture (Lec) and lab (Lab) sessions.

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