New Enzymes by Directed Evolution
This course aims to experience different technologies that are used during protein engineering experiments. These technologies will be explained in detail in theory and in practice with the idea that the student can apply them independently during the course project and in future research.
Discover and apply a range of technologies and techniques to generate, isolate, and enhance mutated bacterial proteins. Lectures and readings about protein structure, function and evolution are complemented by an extensive laboratory project in directed protein evolution that develops applicable research skills. Special topics in protein engineering include gene mutations, ancestral protein reconstruction, and rational design of new enzymes. Explore additional topics by journal club and student presentations.
1. Intro course/Recombinant DNA technology (Lab: Medium preparation, transformation for overexpression of plasmid)
2. (Lab: Plasmid extraction, quantification (4hrs))
3. Introduction on protein dynamics (1.5hr) (Lab: PCR gene, DNA gel, DNA purification (6hrs))
4. Journal club (1h) (Lab: Library generation by error prone PCR, DNA isolation (6hrs))
5. Alpha Fold (1.5hr)(Lab: Digestion insert and vector, purification, set up ligation (8hrs))
6. Journal club (1h)(Lab: Medium preparation, EtOH purification and transformation in expressing bacterial strains (BL21DE3). (5hrs))
7. Ancestral sequence reconstruction (Dr Clifton) (1.5hr) (Lab: Colony PCR, DNA isolation (5hrs)
8. Journal club (1h)(Lab: Sager sequencing preparation, Sager sequencing (8hrs))
9. Enzyme catalysis(1.5) (Lab: I Round of selection: Preparation of Kan Plate (different conc), Transformation (6hrs))
10. Journal club (1h)(Lab: colony PCR of the surviving colonies and plasmid isolation (5hrs))
11. Neutral mutations, deleterious mutations, epistatic mutations (1.5hr) (Lab: II Round of selection: Preparation of Kan Plate (different conc), Transformation (6hrs))
12. Journal club (1h) (Lab: Colony PCR of the surviving colonies and plasmid isolation (5hrs))
13. Design of an improved enzyme by directed evolution experiments (1.5hr) (Lab: Extra time to repeat or catch up with experiments)
14. Journal club (1h) (Lab: Extra time to repeat or catch up with experiments)
15. Journal club (1h)
16. Final presentation (two students) (Lab: Presentation of a research article where they perform Directed Evolution)
17. Final presentation (two students) (Lab: Presentation of a research article where they perform Directed Evolution)
Homework: 25%, Laboratory report 50%, Presentation, 25%.
Undergraduate level biochemistry or molecular biology
1. Structure and Mechanism in protein Science. A Guide to Enzyme Catalysis and Protein Folding. Alan Fersht, World Scientific.
2. Introduction to Proteins. Structure, Function, and Motion, Second Edition. By Amit Kessel, Nir Ben-Tal
Enrollment cap of 8