Molecular Biology and Gene Technology II

Data

Official data in SubjectManager for the following academic year: 2024-2025

Topic

Course lectures present the theoretic background and practical utilization of current and future-proof methodology of molecular biology, and within that genetic engineering. The course includes the following methodologies: various PCR types, restriction endonuclease digestion, molecular cloning, RNA interference and CRISPR/Cas9, transduction and transfection of pro- and eukaryotic cells, expression systems, development of genetically modified cells and organisms, gene therapy applications. During course seminars the students will familiarize with basic molecular biology and within that genetic engineering-related trouble-shooting. During course practices pipetting and precision skills, sterile techniques, master-mix preparation, serial dilutions etc. will be practiced. Solving simple practical issues will help to gain deep understanding and practical knowledge. Having fulfilled this molecular biology and genetic engineering-based course the graduates will get hands-on- expertise that will aid their efforts on the labor-market for both academic and industrial positions.

Lectures

• 6. Eukaryotic transient transfection (chemical) - Kvell Krisztián
• 5. Eukaryotic transient transfection (physical) - Kvell Krisztián
• 4. Plasmid subtypes used for genetic engineering - Kvell Krisztián
• 3. Plasmids as vaults of genetic information - Kvell Krisztián
• 2. Molecular cloning methods - Kvell Krisztián
• 1. Molecular cloning as tool for gain/loss of function studies - Kvell Krisztián
• 7. Eukaryotic stable transfection (resistance gene) - Kvell Krisztián
• 8. Eukaryotic stable transfection (marker gene) - Kvell Krisztián
• 9. Evaluation of transfection (microscope visualization) - Kvell Krisztián
• 10. Evaluation of transfection (flow-cytometry) - Kvell Krisztián
• 11. Evaluation of transgene function (gain of function) - Kvell Krisztián
• 12. Evaluation of transgene function (loss of function) - Kvell Krisztián
• 13. Conventional methods of GM animal production - Kvell Krisztián
• 14. Fast methods of GM animal production - Kvell Krisztián
• 15. Consultation - Kvell Krisztián
• 16. Midterm evaluation - Kvell Krisztián
• 17. RNA interference - Kvell Krisztián
• 18. RNAi toolbox: siRNA, miRNA, mimic - Kvell Krisztián
• 19. Neglected DNA editing tool I: zinc finger domains - Kvell Krisztián
• 20. Neglected DNA editing tool II: sleeping beauty - Kvell Krisztián
• 21. DNA editing function of CRISPR in eukaryotes - Kvell Krisztián
• 22. Natural function of CRISPR in prokaryotes - Kvell Krisztián
• 23. Ribozymes as dual role biological macromolecules - Kvell Krisztián
• 24. Artificial ribozymes - Kvell Krisztián
• 25. Shadow biosphere concept - Kvell Krisztián
• 26. Harnessing environmental bacterial strains - Kvell Krisztián
• 27. Consultation - Kvell Krisztián
• 28. Final evaluation - Kvell Krisztián

Practices

• 6. Performing Ca2PO4 transfection on human cells - Kvell Krisztián
• 5. Performing lipofection on human cells - Kvell Krisztián
• 4. Ligation of new plasmid construct - Kvell Krisztián
• 3. Restriction digestion of donor and host plasmid - Kvell Krisztián
• 2. Purification and ligation of gene of interest - Kvell Krisztián
• 1. Cloning gene of interest using Hi-Fi enzymes - Kvell Krisztián
• 7. Performing spinoculation on human cells - Kvell Krisztián
• 8. Applying selection pressure and follow-up - Kvell Krisztián
• 9. Evaluating marker gene expression by microscope - Kvell Krisztián
• 10. Evaluating marker gene expression by flow-cytometry - Kvell Krisztián
• 11. Performing gain of function functional assay - Kvell Krisztián
• 12. Performing loss of function functional assay - Kvell Krisztián
• 13. Blood sample draw from GFP+ mouse strain - Kvell Krisztián
• 14. Blood sample visualization from GFP+ mouse strain - Kvell Krisztián
• 15. Consultation - Kvell Krisztián
• 16. Consultation - Kvell Krisztián
• 17. Performing siRNA lipofection on human cells - Kvell Krisztián
• 18. Visual evaluation of siRNA uptake - Kvell Krisztián
• 19. Performing antisense oligo lipofection on human cells - Kvell Krisztián
• 20. Gene expression evaluation of antisense oligo effect - Kvell Krisztián
• 21. Performing CRISPR lipofection on human cells - Kvell Krisztián
• 22. Gene expression evaluation of CRISPR efficiency - Kvell Krisztián
• 23. Performing quantum biology enzymatic experiment - Kvell Krisztián
• 24. Evaluating quantum biology enzymatic experiment - Kvell Krisztián
• 25. Plating environmental bacterial strains - Kvell Krisztián
• 26. Observing differences between environmental strains - Kvell Krisztián
• 27. Consultation - Kvell Krisztián
• 28. Final evaluation - Kvell Krisztián

Seminars

• 3. Planning lipofection and Ca2PO4 transfection - Kvell Krisztián
• 2. Shuffling gene of interest between plasmids - Kvell Krisztián
• 1. Planning molecular cloning - Kvell Krisztián
• 4. Planning viral vectors - Kvell Krisztián
• 5. Planning visualization experiments - Kvell Krisztián
• 6. Planning functional assays - Kvell Krisztián
• 7. Demonstration of GFP+ mouse strain - Kvell Krisztián
• 8. Consultation - Kvell Krisztián
• 9. Planning RNAi oligos and constructs - Kvell Krisztián
• 10. Planning zinc finger domain DNA editing tool - Kvell Krisztián
• 11. Planning functional assays - Kvell Krisztián
• 12. Enzyme quantum biotechnology concepts - Kvell Krisztián
• 13. Planning experiments with environmental strains - Kvell Krisztián
• 14. Consultation - Kvell Krisztián

Reading material

Obligatory literature

Cox, Doudna, O’Donnel: Molecular Biology: Principles and Practice, Freeman, 2nd Edition, 2015, ISBN:1464126143

Mitra, Sandhya: Genetic Engineering: Principles and Practice, MC GRAW HILL INDIA, 2015, ISBN-10 : 9789339203535, ISBN-13 : 978-9339203535

Literature developed by the Department

Notes

Recommended literature

Krebs, Goldstein, Kilpatrick: Lewin’s Essential Genes, Jones and Bartlett, 4th Edition, 2020.

ISBN:1284173135

Conditions for acceptance of the semester

Labnotes submitted by end of semester

Mid-term exams

Single choice questions prepared according to the subject of the lectures. The written exam test might contain minimum requirement questions where the wrong answers could mean failure on the exam.

Making up for missed classes

Not possible.

Exam topics/questions

https://gytk.pte.hu/en/menupont/10537

Examiners

• Kvell Krisztián

Instructor / tutor of practices and seminars

• Hairui Ou
• Nemes Balázs