1.2.2: Module 4 and 5 Overview
- Page ID
- 25816
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\(\newcommand{\avec}{\mathbf a}\) \(\newcommand{\bvec}{\mathbf b}\) \(\newcommand{\cvec}{\mathbf c}\) \(\newcommand{\dvec}{\mathbf d}\) \(\newcommand{\dtil}{\widetilde{\mathbf d}}\) \(\newcommand{\evec}{\mathbf e}\) \(\newcommand{\fvec}{\mathbf f}\) \(\newcommand{\nvec}{\mathbf n}\) \(\newcommand{\pvec}{\mathbf p}\) \(\newcommand{\qvec}{\mathbf q}\) \(\newcommand{\svec}{\mathbf s}\) \(\newcommand{\tvec}{\mathbf t}\) \(\newcommand{\uvec}{\mathbf u}\) \(\newcommand{\vvec}{\mathbf v}\) \(\newcommand{\wvec}{\mathbf w}\) \(\newcommand{\xvec}{\mathbf x}\) \(\newcommand{\yvec}{\mathbf y}\) \(\newcommand{\zvec}{\mathbf z}\) \(\newcommand{\rvec}{\mathbf r}\) \(\newcommand{\mvec}{\mathbf m}\) \(\newcommand{\zerovec}{\mathbf 0}\) \(\newcommand{\onevec}{\mathbf 1}\) \(\newcommand{\real}{\mathbb R}\) \(\newcommand{\twovec}[2]{\left[\begin{array}{r}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\ctwovec}[2]{\left[\begin{array}{c}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\threevec}[3]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\cthreevec}[3]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\fourvec}[4]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\cfourvec}[4]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\fivevec}[5]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\cfivevec}[5]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\mattwo}[4]{\left[\begin{array}{rr}#1 \amp #2 \\ #3 \amp #4 \\ \end{array}\right]}\) \(\newcommand{\laspan}[1]{\text{Span}\{#1\}}\) \(\newcommand{\bcal}{\cal B}\) \(\newcommand{\ccal}{\cal C}\) \(\newcommand{\scal}{\cal S}\) \(\newcommand{\wcal}{\cal W}\) \(\newcommand{\ecal}{\cal E}\) \(\newcommand{\coords}[2]{\left\{#1\right\}_{#2}}\) \(\newcommand{\gray}[1]{\color{gray}{#1}}\) \(\newcommand{\lgray}[1]{\color{lightgray}{#1}}\) \(\newcommand{\rank}{\operatorname{rank}}\) \(\newcommand{\row}{\text{Row}}\) \(\newcommand{\col}{\text{Col}}\) \(\renewcommand{\row}{\text{Row}}\) \(\newcommand{\nul}{\text{Nul}}\) \(\newcommand{\var}{\text{Var}}\) \(\newcommand{\corr}{\text{corr}}\) \(\newcommand{\len}[1]{\left|#1\right|}\) \(\newcommand{\bbar}{\overline{\bvec}}\) \(\newcommand{\bhat}{\widehat{\bvec}}\) \(\newcommand{\bperp}{\bvec^\perp}\) \(\newcommand{\xhat}{\widehat{\xvec}}\) \(\newcommand{\vhat}{\widehat{\vvec}}\) \(\newcommand{\uhat}{\widehat{\uvec}}\) \(\newcommand{\what}{\widehat{\wvec}}\) \(\newcommand{\Sighat}{\widehat{\Sigma}}\) \(\newcommand{\lt}{<}\) \(\newcommand{\gt}{>}\) \(\newcommand{\amp}{&}\) \(\definecolor{fillinmathshade}{gray}{0.9}\)Gleevec is a blockbuster small molecule drug for chronic myelogenous leukemia (CML) that functions as a potent inhibitor of Bcr-Abl, an aberrant kinase implicated in the disease. While most of patients treated with Gleevec in the chronic stage of CML experience remission, a significant population eventually develops resistance to the drug. A number of point mutations in the gene that encodes the Bcr-Abl protein have been identified in patients with Gleevec-resistant CML. Over the semester you will develop and execute a research plan to 1) determine whether a selected mutation (H396P) in the BCR-ABL gene confers Gleevec resistance using an in-vitro kinase assay, 2) explore the efficacy of an alternative Bcr-Abl inhibitor, Dasatinib, on the wild-type and mutant kinases, 3) evaluate crystal structures to understand the mechanism(s) by which Bcr-Abl mutations block drug activity, and 4) use site-directed mutagenesis to create an another Gleevec-resistant Abl mutant of your choice. A brief description of the 15 lab sessions is provided below.
| H396P Abl protein expression/ kinase inhibition assays | DNA site-directed mutagenesis | |
| Session 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. |
| Session 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. |
| Session 3 | Digest isolated DNA to check for the wt Abl insert. Run DNA agarose gel. Design primers for an Abl kinase domain mutant. | |
| Session 4 | Prepare protein purification buffers. Create a BSA standard curve for future protein quantification. | |
| Session 5 | Lyse cells and isolate the H396P Abl kinase domain. Dialyze protein into TBS. | |
| Session 6 | Prepare an SDS-PAGE protein gel. | |
| Session 7/8 | Run SDS protein gel. Concentrate protein and quantify final protein concentration. | |
| Session 9 | Set up PCR for DNA mutagenesis. | |
| Session 10 | Complete the DPN digest and transform storage cells with mutant DNA. Pour LB/agar plates. | |
| Session 11 | Isolate (by miniprep) and quantify DNA. Prepare mutant DNA samples for sequencing. | |
| Session 12 | Prepare buffers and reagents for the coupled kinase activity assay. | |
| Session 13 & 14 | Complete kinase assays: wt Abl kinase domain and the H396P mutant domain in the absence and presence of inhibitors. | |
| Session 15 | Complete crystal structure viewing exercises. | Analyze DNA sequencing results. |