Learning outcomes of the course unit
Upon completion of the course, the student should have gained insight into the main biochemical laboratory techniques. He\she should be able to address simple biochemical problems and analyze simple instrumental data.
1. Knowledge and understanding. At the end of the course, the student should be able to:
- Know and understand the main biochemical methodologies described in the course
- Know and understand the principles of each methodology in molecular details
- Know the applications of the techniques described in the course
2. Applying knowledge and understanding. At the end of the course, the student should be able to:
- Identify the most suitable technique to obtain functional or structural pieces of information on macromolecules
- solve simple problems on the application of the techniques
3. Communication skills. At the end of the course, the student should be able to:
- Discuss the topics described in the course in a clear, concise, and effective way.
- inform the experimental scientific results
4. Learning skills. At the end of the course, the student should be able to:
- make connections between the various topics of the course
- Understand the potential of research in biochemistry.
Basic knowledge of biology and organic chemistry. The teaching module is carried out after the Biochemistry module that should be attended by the student.
Course contents summary
The course will cover the main recombinant DNA techniques and the methods used for the expression in recombinant form, the purification and characterization of proteins.
PART I: RECOMBINANT DNA AND ITS APPLICATIONS
-Recombinant DNA technology: restriction endonucleases, DNA ligase, cloning vectors, plasmids as cloning vectors, host organisms, bacterial cultures, selection of recombinant clones, -complementation, PCR, RT-PCR, site-directed mutagenesis, genomic and cDNA libraries. DNA sequencing
- Expression of recombinant proteins: expression vectors, cell strains, lac and T7 promoters, fusion proteins. Protein therapeutics: insulin and recombinant vaccines.
PART II: PURIFICATION OF PROTEINS AND ELECTROPHORESIS
- Protein purification: extraction techniques; centrifugation; ultrafiltration, dialysis, diafiltration; fractionation methods; chromatography (hydrophobic interaction, ion exchange, size exclusion, affinity).
-Electrophoretic techniques: principles and applications to biological macromolecules. Protein electrophoresis (native, SDS-PAGE and their applications). Two-dimensional electrophoresis. DNA electrophoresis and restriction analysis with its applications).
PART III: CHARACTERIZATION AND IDENTIFICATION OF PROTEINS
- Absorption spectroscopy: principles and instrumentation. Lambert-Beer law. Chromophores of biological interest. Protein absorption spectra. Determination of protein concentration.
- Fluorescence emission spectroscopy: principles (fluorescence emission, Stokes shift, fluorescence quantum yield), instrumentation and applications (fluorophores of biological interest).
-Circular dichroism: principles (linearly polarized radiation, circularly polarized radiation, eliptically polarized radiation and elipticity). Applications to the study of protein structure
- Enzymatic activity assays: continuous methods and discontinuous methods. Direct, indirect and coupled assays. Chromogenic substrates.
- Immunochemistry: antibodies (structure, specificity, labeling). Production of monoclonal and polyclonal antibodies. Immunoassays: RIA and ELISA. Applications: pregnancy tests and tests for the identification of viral infections (HIV and Covid-19). Blotting techniques
- introduction to proteomics
- Metodologie Biochimiche. A cura di Carmela Bonaccorsi di Patti, Roberto Contestabile e Martino Luigi di Salvo. Zanichelli
- Metodologie Biochimiche e Biomolecolari. Mauro Maccarrone. Zanichelli
The textbooks listed above are available only in the Italian version.
- Principles and Techniques of Biochemistry and Molecular Biology. Wilson & Walker. Cambridge University Press.
The teaching activities will be carried out in the form of in-person lectures, with short classroom exercises aimed at the student's self-assessment. Alternative online lectures will depend on decisions from the University.
The teaching activities will be carried out through lectures with the help of slides that will be made available weekly to the students on the Elly website.
Lessons will be integrated with classroom exercises aimed at improving the student's ability to apply theoretical knowledge to real-life problems.
The teacher will show, when possible, videos of the techniques treated in the course, in order to provide students with practical indications useful for understanding the applications of the methods.
Assessment methods and criteria
The exam is oral and takes place in the same session as the Biochemistry exam. The examination will be carried out in person, if allowed by the regulations for the containment of the Covid-19 pandemic.
Questions will cover the entire program and the student will also be asked to solve a problem.
The scoring, on a 18-30 scale, will take into account the level of analysis, the ability to critically apply the knowledge, the appropriateness of the scientific language and the autonomy in the discussion.
Students diagnosed with specific learning disorders (SLD), certified in accordance with law No. 170/2010, will be able to take the exams in the forms required by law.