SUBJECT

Gene Technology and Protein Engineering I.

lecture

master

Faculty of Science

Biology MSc

3

Semester 1

Autumn semester

1. Terminology, historical milestones. Basic principles of molecular cloning. Vectors and inserts, ligation, amplification, selection

2. DNA modifying enzymes, types and applications

3. Basic methods. Design of recombinant constructions.Introduction of DNA into host cells: transformation (transfection) infection, electroporation Electrophoresis, hybridization techniques, DNA chip technology

4. Polymerization chain reaction. Basic principles, enzymes used in PCR, optimalization, primer design. Applications in basic and applied research. Non-PCR based amplification methods

5. Vector-host systems.Prokaryotic and eukaryotic systems, E.coli strains, Plasmid and phagemid vectors, lambda bacteriophage vetors, cosmids, PAC, BAC, shuttle vectors. Yeast and other eukaryotic vectors, artificial chromosomes

6. Recombinant DNA libraries. Construction of genomic and cDNA libraries. Screening methods.

7. DNA sequencing. Sanger’s enzymatic method, fluorescent automatic sequencing, high-through-put sequencing methods

8. Genome projects. Mapping and shot-gun strategies.

9. Introduction into bioinformatics. Most important data bases, sequence analysis, alignments, motifs, predictions

10. In vitro mutagenesis. Random mutagenesis. Site directed mutagenesis, Kunkel method, megaprimer and other methods

11. Expression systems in prokaryotes. Prokaryotic expression vectors. Overexpression systems. Tags and fusion constructs. Isolation of recombinant proteins. In vitro translation systems

12. Expression systems in eukaryotes. Yeast, baculovirus, insect cell expression systems. Mammalian expression systems. Cell cultures, transfection methods, transient or stable expression

13. Methods of gene targeting. Knock-out and knock-in techniques.Gene silencing, RNA interference. DNA manipulations in cloning of eukaryotic organisms

14. Transgenic organisms. Transgenic mouse, gene transfer into ES cells. Transgenic plants

15. Interaction techniques - molecular evolution. Phege and bacterial display systems. Two (and three) hybride systems in yeast and othe cells.

• Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K. and Walter, P.: Molecular Biology of the Cell, Taylor & Francis, 2014, ISBN 9780815344322

• Darnell, J., Lodish, H. and Baltimore D.: Molecular Cell Biology, W. H. Freeman, 2012, ISBN 9781429234139

• Nelson & Cox: Lehninger Principles of Biochemistry – 6th Edition

• W.H. Freeman, 2013; ISBN 9781464109621

• Voet et al: Fundamentals of Biochemistry – 2nd Edition

• Wiley, 2012; ISBN: 9780470547847

• Barnum: Biotechnology – 2nd Edition

• Thomson Learning, 2005; ISBN: 9780495112051