Instructor:
Dr. Joseph J. Provost Office: 208 Science Lab
Telephone:
477-4323 email:
provost@mnstate.edu
Syllabus
| This semester will consist of a research style project.
Unlike
Biol/Chem 405, with guidance from the instructor, you will be designing
most of your experiments. The semester will focus on analysis of
the structure and function of malate dehydrogenase, creating mutations
in the gene, expression and purification of the fusion protein and
determining
the kinetic effects of both the wild type and mutant enzyme. In
groups
of one, two or three you will learn how to mine and work with protien
bioinformatics, use protein structural rendering programs, search
biochemical liturature to understand structure function of a protien,
design a hypothesis, plan all of your experiments, make your
protocols and interpret your
data. Designing the laboratory in this manner is meant to give each student a feel for what a realistic project might be in a research, industrial or pharmaceutical laboratory. |
Homework / Assignment Description and Deadlines
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| Phase I Jan 15- Feb 5 Bioinformatics, Structure & MDH Background |
Dr. Provost's MDH Safari Guide (how to hunt the elusive MDH!) - This document will help tell you what is expected for the semester. Modeling workshop handout and homework - Due Feb 8 - Turn into the chemistry office. NEW Jan 08 - if a file is marked as working on it, skip it until it is marked fixed. All others should still work or are fixed.
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Phase II
Feb 12 Hypothesis & Experimental Design |
Each group will meet
with instructor to work on your hypothesis and experimental
design. Each group WILL have
their hypothesis and a fairly detailed experimental plan ready to
discuss on this day. Required In-Class Day Possible experiments - Additional possibilitys include: - Rapid Protein Folding Assay using GFP - pyruvate, lactate or other carboxylic acid as an inhibitor - heat shock protein interactions List of wild-type and mutuant MDH clones - Temp version for you to work with. Short list of MDH publications Someone find the MDH - LDH papers referenced in the Goward review - first two papers (PDF file only) get extra credit!
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Phase II
Feb 19 Mini Presentation – plan and proposal of work |
In class presentation
and student evaluation of hypothesis, reasoning and experimental
design. Required In-Class Day
(50 pts) |
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Phase
III
Feb 26- 18 March Expression and Purification of MDH
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Work on the purification of each
protein for your project. Depending on your experimental designs,
a Ni+ affinity purification will do just fine. 50 ml Expression Protocol Transformation Protocol - look for this protocol at the bottom of the linked page Lab Materials Instructions and Location List Malate Dehydrogenase Assay Protocol Citrate Synthase Assay Protocol Enzyme Assay Guide |
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Phase
III
18 March – 29 April Enxymatic and Structural Analsysis
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Work on your experiments |
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12:00
May 2
Final Presentations
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Each group will present their
project as a formal powerpoint presentation. - 12 min presentations with 3 min for questions - All members must be equal participants - a CD of the presentation (well labeled) must be turned in at the end of the presentation with the lab book for final grade - Each presentation must include the following: * Intro to MDH, or if doing a technique, a good background on the technique (MUST be detailed and chemical in nature) * You must include some structural component to your talk. The first 30% of the semester was spent on bioinformatics and your project came in part from this. A simple image of the protein alone will not be enough. You learned how to do a number of things with the structure of proteins. Use those skills as it applies to your project and it's hypothesis. * Hypothesis and why or how you came up with the hypothesis. Rember your project was based on papers not an idea alone. No consumer science, use the scientific method. * Methods, Results and Conclusions. |
The final presentations will go longer than
5:00: - Plan to stay late, I will
provide pizza and pop.
IMPORTANT NOTE:
Please use 50 to 100 ml cultures for expression
of your protein. The watermelon MDH is AMP resistant and the
others
are all Kan AND chlorampheticol resistant (there is 1000x concentrated
stock in the freezer). Use the Qiagen protocol for expression of
EACH isozyme. Just use the appropriate antibiotic(s) for your
clone.
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Pages 18 - 2- > Read the info about NTA Pages 48 - 53 -> Expression and Purification info Page 61 Protocol 7 Growth of culture (100 ml) Pages 63 - 75 Purification and buffer Page 79 Protocol 9 Native Lysate Preparation Page 82 Protocol 12 Batch Purification - Native Conditions |
Laboratory Notebooks:
- Successful completion of the work for each of the four main blocks will be worth 100 points.
- The ability to interpret the biochemical literature will be assessed and worth 100 pts. This will be determined by visits with your instructor.
- There will be several assignments with various points.
- At the end of each block you will turn your lab book and any assigned homework (50 points each).
- At the end of the semester each group will present the project using a power point presentation. The presentation will be worth 100 points.
- Student peer evaluation will be worth 25 points.
Lab notebooks and assignments will be turned into the basket in the Biology Office.
For this lab you will need to have a NEW bound ruled notebook.A spiral bound book will NOT suffice.The pages must be numbered, no pages can be removed and the book will be dedicated ONLY for the lab book.Laboratory notebooks are not to be works of art but they must be neat enough to be legible.Use Pen only,Pencil tends to fade with time and many entries can be lost when small spills smear notes taken with pencil or water soluble marker pens.The first two pages would be left for the table of contents.The lab book should have the same organization as last semester.All data will be included in the notebook.The best way to keep up with the lab book is to take the time before to write the purpose of each day and the procedure.Then enter your calculations as you go. Include the observations and data on the fly.
DO NOT WAIT TO ENTER IT IN LATER. Later comes at the end of the semester and is never what it should be. I reserve the right to spot inspect the book to see that you are keeping up on it. If I do so, there will be points assigned as part of the lab book for that block.
MAIN POINT FOR THE LAB BOOK:I want to be able to read this and repeat what you did! No guessing should be needed to repeat your work. That is the true measure of a lab book.These should have sufficient information as to what you are doing and why.Including the preparation steps.