Course program
The course consists of two integrated modules (Molecular Biology Techniques and Protein Purification and Characterization). The program of the course has to be considered comprehensive of both modules.
The course will be divided into two parts: a theoretical section and an experimental section. The first part, which will be held in classroom with theoretical lectures will be devoted to the study of the main methodologies and techniques used in the laboratories of biochemistry and molecular biology. In the second part, which will be held in a devoted laboratory, experiments will be carried out that will cover the topics thought in classroom.
Topics of in class lectures:
• General approach to biochemical research. The philosophy behind the experiment. Inductive and deductive scientific method. Top-down e bottom-up approaches. The holism-reductionism dichotomy. How to choose
an experimental approach. The set up of an experiment.
• Buffer systems. Solutions. Dilutions.
• In vivo and in vitro models.
• Precision and accuracy. The experimental errors and the mistakes. Statistics and indetermination. Analytical vs. preparative techniques.
• Bacterial and eukaryotic cell cultures.
• Recombinant DNA methodologies. Primer design, PCR, restrictions, ligations, competent cell transformation.
• Plasmid DNA extraction from bacterial cultures. Extraction of genomic DNA .
• RNA extraction. RNA reverse-transcription (cDNA). Transcription levels analysis.
• Gene editing by CRISPR/Cas9 technique.
• Biomarkers. Southern, Northern, Western blot. Real time-PCR.
• Immunochemistry. ELISA and immunohistochemical assays.
• Methodologies for the expression of recombinant proteins.
• Fractionation and purification of cellular macromolecules.
• Centrifugation techniques and methods based on solubility.
• Chromatographic and electrophoretic techniques.
• Enzyme kinetics. Enzyme assays. Biotechnological application of enzymatic techniques.
• Structure and characterization of proteins. Interaction between macromolecules. Protein-ligand binding.
• Spectrophotometric techniques, circular dichroism and calorimetry. Dissociation constants measurement.
• Protein crystallography.
• Use of Internet in biochemical research.
Topics of laboratory experiences: the lab experiences may vary slightly from the diagram below, depending on the experimental outcomes.
• Using the internet in biochemical research.
• Cloning of cDNA into expression vectors.
• Purification of recombinant proteins.
• Structural and functional characterization of a purified protein
• Gene targeting by CRISPR/Cas9 (Design of constructs by online software; preparation of constructs and directional cloning into vectors; analysis of recombinant vectors)
Laboratory classes have to be considered mandatory
Prerequisites
1) ESSENTIAL: students must have followed the courses of Bioorganic Chemistry (I semester) e Structure and Function of Macromolecules (I semester); the student should ideally have passed the related exams;
2) ESSENTIAL: availability for the mandatory practical laboratory experiences and aptitude for group work;
3) ESSENTIAL: knowledge of the basic concepts of Biochemistry, and Molecular and Cell Biology. In particular, it is necessary to know: a) concepts and properties of buffer solutions, solution theory and stoichiometric calculation for their preparation; b) basic concept of cellular physiology and cell cultivation techniques; c) structural and functional characteristics of DNA, with particular regard to plasmid vectors used in recombinant DNA techniques; d) structural and functional characteristics of proteins with particular attention to enzymes;
3) IMPORTANT: knowledge of the basic technologies used in molecular biology and biochemistry, including recombinant DNA methodologies, chromatography, electrophoresis, and enzymatic assays;
4) IMPORTANT: basic knowledge of databases for search of scientific literature, protein and nucleotides sequences.
Books
• A general Biochemistry textbook (e.g. Voet&Voet, Leningher, Stryer) and a Molecular Biology textbook (e.g. Watson, Lewin) should be available for consultation at any time.
• Analytical Techniques in Biochemistry and Molecular Biology, by Rajan Katoch - ISBN 978-1-4419-9784-5, e-ISBN 978-1-4419-9785-2, DOI 10.1007/978-1-4419-9785-2 – Springer.
• Biochemical Methods: A Concise Guide for Students and Researchers, by Alfred Pingoud, Claus Urbanke, Jim Hoggett, Albert Jeltsch – Wiley. ISBN: 978-3-527-30299-4, October 2002, 374 Pages.
• Biochemical Calculations: How to Solve Mathematical Problems in General Biochemistry, by Irwin H. Segel - Publisher: Wiley, 458 pagine, ISBN-10 : 9788126526437.
• Gene Cloning and DNA Analysis: An Introduction, 8th Edition, by T. A. Brown – Publisher: Wiley-Blackwell, 432 pages, ISBN: 978-1-119-64067-7
• PowerPoint lessons will be available - Lecture and laboratory notes will also be made available.
• Students, divided in small working groups, will be responsible for keeping a laboratory notebook updated with all the experiments that will be carried out, methodologies, design of experiments, data and discussion of
results.
Teaching mode
The course consists of two integrated modules (Molecular Biology Techniques and Protein Purification and Characterization). The layout of the teaching activities has to be considered comprehensive of both modules.
The course will be carried out through theoretical lectures (32 hours), theoretical classroom exercises (12 hours) and practical laboratory experiences (48 hours).
Lectures (32 hours):
• Explanation of the topics covered by the program through slides and audiovisual material. This educational model is aimed at providing the theoretical knowledge of most common biochemical methodologies.
• Open discussion of the topics of the lessons, during which the students are called to intervene, which have the purpose of developing the skills of communication, criticism and judgment.
Theoretical exercises in class (12 hours):
• Stoichiometry exercises on the preparation of buffer solutions and sample dilutions (2 hours). Particular attention will be paid to the understanding of the importance of buffer solutions and to the preparation of
solutions in general
• Exercises on nucleotide and protein sequences retrieving from databases (4 hours)
• Exercises on cloning strategies of cDNA fragments into vectors (4 hours)
• Compilation of a purification table (2 hours)
Laboratory experiences (48 hours):
• Plasmid DNA purification, digestion with restriction enzymes and control on analytical gel (4 hours)
• Preparation of culture media and of bacterial competent cells for plasmid trasformation (4 hours)
• CRISPR/Cas9 vector digestion and agarose gel analysis (3 hours)
• Construct preparation by oligonucleotide annealing and 5’-end enzymatic phosphorylation (3 hours)
• Ligation to join DNA constructs and the digested vector, competent bacteria transformation (4 hours)
• Screening of recombinant clones by colony PCR followed by separation on agarose gel (5 hours)
• Preparation of vector DNA from positive clones (5 hours)
• Bacterial growth, expression and purification of a recombinant protein, and analysis on SDS-PAGE (8 hours)
• Determination of protein concentration in solution and set up of a crystallization plate (3 hours)
• Enzymatic kinetic measurements (4 hours)
• Preparation of the apo form of an enzyme (3 hours)
• Measurement of a protein-ligand dissociation constant (2 hours)
The laboratory experiences, which are mandatory, are designed to put into practice the theoretical concepts studied in class, to develop the ability to plan an experiment and interpret it, to increase critical and judgmental skills.
Frequency
Not mandatory
Exam mode
The course consists of two integrated modules (Molecular Biology Techniques and Protein Purification and Characterization). The exam will be administered as one, comprehensive of both modules.
EXAM PROCEDURE: During the course, students will be divided into groups of 3-4 people. Each group will always work together during the lab experiences and will have to keep a lab notebook with all the experiments that will be performed. Each group will then write a laboratory report in which the various experiences are elaborated, described and discussed, that must be handed to the teachers one week before the exam. The final exam will include the discussion of this report using a PowerPoint presentation prepared by each individual group.
In general, the student's preparation will be evaluated on the basis of his ability to describe the experiments performed and the relative methodologies in a clear and scientifically rigorous manner and to know how to link the various topics, showing that he understood the methodological rationale. For the purposes of the overall assessment of the student's preparation, the skills of communication, criticism and judgment will also be taken into consideration.
Lesson mode
The course consists of two integrated modules (Molecular Biology Techniques and Protein Purification and Characterization). The layout of the teaching activities has to be considered comprehensive of both modules.
The course will be carried out through theoretical lectures (32 hours), theoretical classroom exercises (12 hours) and practical laboratory experiences (48 hours).
Lectures (32 hours):
• Explanation of the topics covered by the program through slides and audiovisual material. This educational model is aimed at providing the theoretical knowledge of most common biochemical methodologies.
• Open discussion of the topics of the lessons, during which the students are called to intervene, which have the purpose of developing the skills of communication, criticism and judgment.
Theoretical exercises in class (12 hours):
• Stoichiometry exercises on the preparation of buffer solutions and sample dilutions (2 hours). Particular attention will be paid to the understanding of the importance of buffer solutions and to the preparation of
solutions in general
• Exercises on nucleotide and protein sequences retrieving from databases (4 hours)
• Exercises on cloning strategies of cDNA fragments into vectors (4 hours)
• Compilation of a purification table (2 hours)
Laboratory experiences (48 hours):
• Plasmid DNA purification, digestion with restriction enzymes and control on analytical gel (4 hours)
• Preparation of culture media and of bacterial competent cells for plasmid trasformation (4 hours)
• CRISPR/Cas9 vector digestion and agarose gel analysis (3 hours)
• Construct preparation by oligonucleotide annealing and 5’-end enzymatic phosphorylation (3 hours)
• Ligation to join DNA constructs and the digested vector, competent bacteria transformation (4 hours)
• Screening of recombinant clones by colony PCR followed by separation on agarose gel (5 hours)
• Preparation of vector DNA from positive clones (5 hours)
• Bacterial growth, expression and purification of a recombinant protein, and analysis on SDS-PAGE (8 hours)
• Determination of protein concentration in solution and set up of a crystallization plate (3 hours)
• Enzymatic kinetic measurements (4 hours)
• Preparation of the apo form of an enzyme (3 hours)
• Measurement of a protein-ligand dissociation constant (2 hours)
The laboratory experiences, which are mandatory, are designed to put into practice the theoretical concepts studied in class, to develop the ability to plan an experiment and interpret it, to increase critical and judgmental skills.
