Course Coordinator: 
Paola Laurino
New Enzymes by Directed Evolution

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. 

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.
Detailed Syllabus: 
  1. Week 1 Recombinant DNA technology: PCR, cloning, site-specific mutagenesis, random mutagenesis (2hrs) 

Lab 1.1 Medium preparation, transformation for overexpression of plasmid (4hrs) 

Lab 1.2 Plasmid extraction, quantification (4hrs) 

  1. Week 2 Introduction on enzymes activity: case study Kan Kinase (1hr) 

Lab 2.2 PCR gene, DNA gel, DNA purification (6hrs) 

Lab 2.3 library generation by error prone PCR, DNA isolation (6hrs) 

  1. Week 3 Neutral mutations, deleterious mutations, epistatic mutations (1hr) 

Lab 3.1 Digestion insert and vector, purification, set up ligation (8hrs) 

Lab 3.2 Medium preparation, EtOH purification and transformation in expressing bacterial strains (BL21DE3). (5hrs) 

  1. Week 4 Sager sequencing introduction (1hr) 

Lab 4.1 Colony PCR, DNA isolation, (5hrs) 

Lab 4.2 Sager sequencing preparation, Sager sequencing (8hrs) 

  1. Week 5 Design of a improved enzyme by directed evolution experiments: kan kinase as case study (1hrs) 

Lab 5.1 I Round of selection: Preparation of Kan Plate (different conc), Transformation (6hrs) 

Lab 5.2 colony PCR of the surviving colonies and plasmid isolation (5hrs) 

  1. Week 6 Protein visualisation programs introduction -pymol (2hrs) 

Lab 6.1 II Round of selection: Preparation of Kan Plate (different conc), Transformation (6hrs) 

Lab 6.2 Colony PCR of the surviving colonies and plasmid isolation (5hrs) 

  1. Lab 7.1 Sager sequencing of a pool of surviving variants (8hrs) 

Lab7.2 Extra time to repeat or catch up with experiments (6hrs) 

  1. Presentation of a research article where they perform Directed Evolution (2-4hrs depending of number of students) 

Course Type: 
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.
Prior Knowledge: 

Undergraduate level biochemistry or molecular biology