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Lecturer(s)
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Zalabák David, Mgr. Ph.D.
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Course content
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1. Introductory lecture. Preparing the bacterial inoculum (E. coli) carrying the expression vector. Preparing cultivation media and sterile glass flasks. 2. Setting up the heterologous protein expression, IPTG induction, preparing buffers, solutions etc. 3. Harvesting bacterial cells, cell lysis and protein purification using affinity chromatography. 4. Dialysis and concentration of protein sample using ultrafiltration. 5. Analysis of protein sample using SDS-PAGE method to test protein purity and molecular weight. Protein quantification using Bradford assay.
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Learning activities and teaching methods
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Dialogic Lecture (Discussion, Dialog, Brainstorming), Demonstration, Laboratory Work
- Attendace
- 39 hours per semester
- Homework for Teaching
- 15 hours per semester
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Learning outcomes
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Main goal of this course is to teach students the techniques of heterologous protein expression. In the framework of this course, students will learn how to prepare and purify recombinant proteins in E. coli, and also how to biochemically characterize them.
Student will apply his/her knowledge of the basic biochemical methods and techniques acquired during his/her former studies. Respective experiments follow sequentially and thus student learns how to plan experimental procedures as well as the time schedule of experiments.
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Prerequisites
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Course is opened for Master students in the 1st and 2nd year.
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Assessment methods and criteria
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Student performance, Systematic Observation of Student
Course will take one full week (7 days), during summer semester. Student should actively participate in the course and to perform all the necessary experiments. Finally, student will write a short report summarizing all the experimental procedure and results.
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Recommended literature
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A guide to Polyacrylamide Gel Electrophoresis and Detection (BioRad) http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6040.pdf.
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PROTEIN EXPRESSION & ANALYSIS IMPACT Kit Instruction Manual (New England Biolab) https://www.neb.com/-/media/catalog/datacards-or-manuals/manuale6901.pdf.
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Kakoli Bose. (2022). Textbook on Cloning, Expression and Purification of Recombinant Proteins.. Springer Singapore.
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Zalabák D., Galuszka P., Mrízová K., Podlešáková K., Gu R., Frébortová J. (2014). Biochemical characterization of the maize cytokinin dehydrogenase family and cytokinin profiling in developing maize plantlets in relation to the expression of cytokinin dehydrogenase genes. Plant Phys. Biochem. 74, 283-293.
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