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Biochemistry Laboratory

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  • Course Description

    The course, which spans two thirds of a semester, provides students with a research-inspired laboratory experience that introduces standard biochemical techniques in the context of investigating a current and exciting research topic, acquired resistance to the cancer drug Gleevec. Techniques include protein expression, purification, and gel analysis, PCR, site-directed mutagenesis, kinase activity assays, and protein structure viewing. This class is part of the new laboratory curriculum in the MIT Department of Chemistry. Undergraduate Research-Inspired Experimental Chemistry Alternatives (URIECA) introduces students to cutting edge research topics in a modular format.

    About Dr. Elizabeth Vogel Taylor

    Dr. Elizabeth Vogel Taylor received her Ph.D. in Organic Chemistry from MIT in the laboratory of Professor Barbara Imperiali of MIT’s Department of Chemistry. Along with HHMI Professor Catherine Drennan, she has been involved in developing and conducting an expanded TA training program for incoming chemistry graduate student TAs at MIT. This 20-hour TA training program focuses on creating a supportive teaching environment and increasing TAs’ ability to lead successful recitation sections. The program has had incredible success in the quality of undergraduate chemistry education and is now a department-wide program, with plans to provide online instructions for nationwide dissemination. In addition, Dr. Taylor is also involved in other HHMI education initiatives headed by Professor Drennan, such as the “Getting Biologists excited about Chemistry” initiative. Under this initiative, Dr. Taylor and Professor Drennan created over 30 succinct (2-5 minutes) modular in-class examples for MIT’s introductory chemistry course (5.111) that relate basic chemistry concepts covered in general chemistry to biology topics. The goal of these examples is to increase students’ interest in chemistry and illustrate how chemistry plays a fundamental role in everyday biology. In addition to being an instructor and course developer for the introductory chemistry course (5.111), Dr. Taylor is teaching the Graduate Students Teaching Certificate program offered by the Teaching & Learning Lab during the academic year 2011-2012.

    Note: Contents for this page are Licensed from http://ocw.mit.edu under the Creative Commons Attribution Share-Alike license.

    School
    Massachusetts Institute of Technology

    Course Code
    5.36

    Date Taught
    Spring/Summer 2009

    Level
    Undergraduate (First Year)
  • Course Meeting Times

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

    Labs: 2 sessions / week, 4 hours / session

    Overview

    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.

    Pre-Labs and Lab Participation

    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.


    SES #

    H396P ABL PROTEIN EXPRESSION/ KINASE INHIBITION ASSAYS

    DNA SITE-DIRECtED MUTAGENESIS

    Lec 1

    Introduction. Kinases in healthy and cancerous cells

    Lab 1

    Grow a starter culture of cells with the H396P Abl and Yop-encoding vectors.

    Grow a starter culture of cells with the wild type Abl vector.

    Lab 2

    Express the H396P Abl protein. (Spin down cells on the following day.)

    Isolate wt-Abl vector DNA through a miniprep. Quantify DNA concentration by UV-Vis.

    Lec 2

    DNA digestion and PCR

    Lab 3

     

    Digest isolated DNA to check for the wt Abl insert. Run DNA agarose gel. Design primers for an Abl kinase domain mutant.

    Lec 3

    Affinity tags for protein purification / detection

    Lab 4

    Prepare protein purification buffers. Create a BSA standard curve for future protein quantification.

     

    Lab 5

    Lyse cells and isolate the H396P Abl kinase domain. Dialyze protein into TBS.

     

    Lab 6

    Prepare an SDS-PAGE protein gel.

     

    Lec 4

    Conserved and variable structural features of kinase domains

    Lab 7 / Lab8

    Run SDS protein gel. Concentrate protein and quantify final protein concentration.

     

    Lec 5

    Site directed mutagenesis (Quickchange method)

    Lab 9

     

    Set up PCR for DNA mutagenesis.

    Lab 10

     

    Digest template DNA, and transform storage cells with mutant DNA. Pour LB/agar plates.

    Lab 11

     

    Isolate (by miniprep) and quantify DNA. Prepare mutant DNA samples for sequencing.

    Lec 6

    Detecting kinase activity

    Lab 12

    Prepare buffers and reagents for the coupled kinase activity assay.

     

    Lab 13 / Lab 14

    Complete kinase assays: wt Abl kinase domain and the H396P mutant domain in the absence and presence of inhibitors.

     

    Lab 15

    Complete crystal structure viewing exercises.

    Analyze DNA sequencing results.

     


  • DescriptionTypeLink
    Complete student manuaDownloadClick
    Supplemental TA instructionsDownloadClick
    Table of contentsDownloadClick
    Equipment and supplies listDownloadClick
    Lab 1DownloadClick
    Lab 2DownloadClick
    Lab 3DownloadClick
    Lab 4DownloadClick
    Lab 5DownloadClick
    Lab 6DownloadClick
    Lab 7DownloadClick
    Lab 8DownloadClick
    Lab 9DownloadClick
    Lab 10DownloadClick
    Lab 11DownloadClick
    Lab 12DownloadClick
    Lab 13DownloadClick
    Lab 15DownloadClick
  • DescriptionTypeLink
    Lec 1 - Introduction. Kinases in healthy and cancerous cellsDownloadClick
    Lec 2 - DNA digestion and PCRDownloadClick
    Lec 3 - Affinity tags for protein purification/detectionDownloadClick
    Lec 4 - Conserved and variable structural features of kinase domainsDownloadClick
    Lec 5 Site directed mutagenesis (Quikchange method)DownloadClick
    Lec 6 - Detecting kinase activityDownloadClick
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