Learning outcomes of the course unit
Learning about fundamental and advanced techniques used for the isolation and determination of biological macomolecules, in particular DNA and proteins.
Course contents summary
1. The biochemical laboratory: chemical risk and biological risk. Cell cultures. Sterilizing materials and ensuring sterile conditions.
2. Recombinant DNA technology: host organisms, restriction endonucleases, DNA ligases, cloning vectors, plasmids as cloning vectors, recombinant selection, alpha-complementation, PCR, site-specific mutagenesis, DNA-microarrays, gene libraries.
3. Recombinant protein expression: parameters affecting the quantity of protein expressed, expression vectors, cell strains, LAC and T7 promoters, expression induction, cell-free expression, fusion proteins. Recombinant proteins as drugs.
4. Protein purification:
* Extraction techniques: mechanical and non-mechanical methods.
* Centrifugation techniques: principles, types of centrifuge, differential sedimentation, gradient centrifugation.
* Solution concentration and pad change: ultrafiltration, dialysis, diafiltration.
* Methods of fractioning: by stability and by solubility (salting out, by pI, and by the addition of organic solvents).
* Chromatography: principles and techniques. Ion exchange chromatography, hydrophobic interaction chromatography, inverse phase chromatography, size exclusion chromatography and its applications, affinity chromatography (IMAC, protein A, GST)
5. Electrophoretic techniques: principles and applications for biological macromolecules. Mobile phase and zone electrophoresis, solid-phase substrates, protein electrophoresis (in native conditions, and SDS-PAGE with applications), protein band detection, Western blotting, isoelectrofocalization, DNA electrophoresis, DNA band detection, an outline of restriction analysis and its applications, Northern blotting, Southern blotting, DNA sequencing.
6. Two-dimensional electrophoresis and mass spectrometry.
7. Spectroscopic techniques:
* Absorption spectroscopy: the Lambert-Beer law, layout of a colorimeter and a spectrophotometer. Protein absorption spectra. Colorimetric assays for protein concentration determination.
* Fluorescence spectroscopy: principles (emission from singlet and triplet state, Stokes shift, quantum fluorescence yield), applications (study of proteic dynamics, collisional quenching, and exposure of fluorophores to solvent, FRET and calcium sensors).
* Circular dichroism: principles (linear radiation polarization, circular radiation polarization, elliptical radiation polarization and ellipticity). Applications in the study of protein structure.
* NMR: an outline of its applications in the study of protein dynamics and structure. Isotopic protein enrichment for expression in minimal media.
8. Enzymatic techniques:
* Studies on stationary state: continuous and discontinuous methods. Direct, indirect and coupled assay. Chromogenic substrates. Radioisotopic methods and product separation.
* Studies on pre-stationary state: rapid mixing methods (stopped-flow) and relaxation methods (pH-jump with caged-H+ compounds).
9. Chemical immune techniques: antibodies (structure, specificity, marking). The production of monoclonal and polyclonal antibodies. Immune dosage levels: RIA and ELISA. Applications: pregnancy test and HIV test.
10. Radioactive isotopes: their use, and measuring radioactivity.
11. Protein crystallization: principles and methods. X-ray diffusion, diffraction maps, electron density maps. Protein data bank: an outline of the use and display of three-dimensional protein structure.
Wilson and Walker: “Biochimica e Biologia Molecolare", new Italian edition. Cortina Editore.
Reed et al.: “Metodologie di base per le scienze biomolecolari”. Zanichelli
T.A. Brown. “Biotecnologie Molecolari: Principi e Tecniche”. Zanichelli
NELSON e COX: “I principi di biochimica di Lehninger”, fourth edition. Chapter 9. Zanichelli