A220
Course Coordinator: 
Paola Laurino
New Enzymes by Directed Evolution
Description: 

During this course the student will experience a research project. We are planning to run enzyme evolution experiments generating a random library of enzyme mutants and selecting for improved activity in vivo (bacterial strains). Student will create variants of Kan Kynase enzymes. The enzymes will be selected for higher antibiotic resistance to assess improved variants. After a few rounds of evolution, advantageous mutations will be enriched in the variants pool and identified by sequencing. The enriched mutations will be highlighted in the protein structure and then analysed. 

Aim: 
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.
Course Content: 
 
 
 
 
 
 

Lecture 
 
 
 
 

Date 
 
 
 
 

Topic 
 
 
 
 

Lab experience 
 
 
 
 

1 
 
 

Sep 26 
 
 

Intro course/Recombinant DNA technology (1.5h) 
 
 

Medium preparation, transformation for overexpression of plasmid  
 
 
 
 

2 
 
 

Sep 27 

 

 
 
 

  

 
 
 

Plasmid extraction, quantification (4hrs)  
 
 
 
 

3 
 
 

Oct 3 

 
 
 

Introduction on protein (dynamics) (1.5hr) 
 
 

PCR gene, DNA gel, DNA purification (6hrs)  
 
 
 
 

4 
 
 

Oct 4 
 
 

Journal club (1h) 

 
 
 

Library generation by error prone PCR, DNA isolation (6hrs)  
 
 
 
 

5 
 
 

Oct 17 
 
 

Alpha Fold (Mr Stefano Pascarelli) (1.5hr)  

 
 
 

Digestion insert and vector, purification, set up ligation (8hrs)  
 
 
 
 

6 
 
 

Oct 18 
 
 

Journal club (1h) 

 
 
 

Medium preparation, EtOH purification and transformation in expressing bacterial strains (BL21DE3). (5hrs)  
 
 
 
 

7 
 
 

Oct 24 
 
 

Ancestral sequence reconstruction (Dr Clifton) (1.5hr)  
 
 

Colony PCR, DNA isolation, (5hrs)  
 
 
 
 

8 
 
 

Oct 25 
 
 

Journal club (1h) 

 
 
 

Sager sequencing preparation, Sager sequencing (8hrs)  
 
 
 
 

9 
 
 

Oct 31 
 
 

Enzyme catalysis (1.5) 
 
 

I Round of selection: Preparation of Kan Plate (different conc), Transformation (6hrs)  
 
 
 
 

10 
 
 

Nov 1 
 
 

Journal club (1h) 

 
 
 

colony PCR of the surviving colonies and plasmid isolation (5hrs)  
 
 
 
 

11 
 
 

Nov 7 
 
 

Neutral mutations, deleterious mutations, epistatic mutations (1.5hr)  
 
 

II Round of selection: Preparation of Kan Plate (different conc), Transformation (6hrs)  
 
 
 
 

12 
 
 

Nov 8 

 

 
 
 

Journal club (1h) 
 
 

Colony PCR of the surviving colonies and plasmid isolation (5hrs) 
 
 
 
 

13 
 
 

Nov 14 

 

 
 
 

Design of an improved enzyme by directed evolution experiments (1.5hr)  
 
 

Extra time to repeat or catch up with experiments  
 
 
 
 

14 
 
 

Nov 15 
 
 

Journal club (1h) 
 
 

Extra time to repeat or catch up with experiments  
 
 
 
 

15 
 
 

Nov 21 
 
 

Journal club (1h) 
 
 

 
 
 
 
 

16 
 
 

Nov 22 
 
 

Final presentation (two students) 
 
 

Presentation of a research article where they perform Directed Evolution  
 
 
 
 

17 
 
 

Nov 28 
 
 

Final presentation (two students) 
 
 

Presentation of a research article where they perform Directed Evolution  
Course Type: 
Elective
Credits: 
2
Assessment: 
Homework: 25%, Laboratory report 50%, Presentation, 25%.
Text Book: 
Structure and Mechanism in Protein Science. A Guide to Enzyme Catalysis and Protein Folding. Alan Fersht, World Scientific.
Introduction to Proteins Structure, Function, and Motion, Second Edition ByAmit Kessel, Nir Ben-Tal
Prior Knowledge: 

Undergraduate level biochemistry or molecular biology