APPLIED BIOCHEMISTRY

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.