Ateneo - UPO

The “Introductory notes” of the “Applied Biochemistry” course illustrate the application of biochemical methods to different fields in the bio-medical and biotechnology research areas; this section also briefly recaps the chemical, physical and functional properties of nucleic acids and proteins that can be exploited for the isolation, the production, the analysis and the manipulation of these fundamental biological macromolecules. The following part is structured in two main teaching modules: the first one describes the recombinant DNA techniques and the systems that can be used to produce recombinant proteins, including the use of publicly accessible databases for the analysis and comparison of nucleotide and amino acid sequences; the second section describes the currently available technology, instrumentation, materials and methods for i) the purification of proteins/macromolecular complexes (from natural sources or expressed in recombinant form), ii) their characterisation, and iii) their modification to perform specific biochemical tasks or to improve their characteristics.

-Stoppini, M e Bellotti, V “Biochimica Applicata” Ed. EdiSES
ISBN: 9788879597135
- Bonaccorsi di Patti, MC, et al. “Metodologie Biochimiche” Ed. CEA
ISBN: 9788808183293
-Amaldi, F et al. "Tecniche e metodi per la biologia molecolare"
ISBN: 9788808920348
- Further selected Reviews and scientific articles (full papers), suggested by the Lecturer.
The presentations used during the lectures will be made available to the students, at the end of each macro-section (i.e. manipulation, analysis and purification of nucleic acids; analysis and purification of proteins; study of interactions between macromolecules); they should be considered as a guide for the detailed study of the same topics on the suggested textbooks.

The "Applied Biochemistry" course describes the theoretical and practical aspects of the study of biological macromolecules, and the impact of the discipline in different fields, ranging from life sciences to analytical applications. The course aims to equip the student with a solid background for her/his future research activities in the protein science area, and the exploitation of the gained knowledge also in the study of other disciplines. To this end, advanced methods for the production, purification, manipulation and biochemical characterisation of macromolecules will be illustrated. Virtual “research projects” will be assigned to small groups of students, aiming at stimulating the student's attitude to an independent and critical thinking and to work in team.

A basic knowledge of the following topics is required:
1) the biochemistry of proteins and nucleic acids
2) the basic processes underlying DNA replication, transcription and protein synthesis, with special emphasis on bacterial systems.

Standard lectures. Audio/video materials (in English) may be used during the lectures.
2-4 hours will be used to allow the presentation of the virtual projects and their peer-to-peer discussion. Final test simulations and training session on problem solving will be regularly scheduled.

Students with physical disabilities, Learning Disabilities or Special Education Needs can request specific services and tools via the Staff Sviluppo e Coordinamento Carriere e Servizi alle Studentesse e agli Studenti, consulting the University webpage: https://www.uniupo.it/en/services/services-students-physical-or-learning-disabilities

Students with disabilities, learning disabilities or special education needs, once they have contacted the University Staff, can refer to the tutor in charge of the course to define the examination modalities, concerning academic aspects.

Written exam.
The first exercise asks the students to design a vector for the expression of a recombinant protein (max. 8 pt); the second exercise consists in the definition of a strategy for the over-expression, the purification and the functional characterisation of a recombinant protein (max. 10 pt); the short assay may relate to a technical subject (e.g. "principles and applications of affinity chromatography") or to broader topics (e.g. "Analysis of macro-molecular interactions by using biophysical methods) (max. 12 pt). To pass the test students must obtain at least the following scores: 1st exercise 5/8; 2nd exercise: 5/10; short assay: 8/12. Up to 3 additional points will be assigned to the members of each team, on the basis of their oral presentation and discussion of the “virtual project”.

The recombinant DNA technology applied to the expression of proteins. The Polymerase Chain Reaction: principles, equipment, primer pairs and specific protocol design. Electrophoresis on agarose gel for DNA analysis: principles and instruments. Sub-cloning vectors: general characteristics. Plasmid vectors for the expression of recombinant proteins in bacteria. Other vectors for the generation of recombinant DNA molecules. Transformation of competent bacteria. Purification of plasmid DNA from bacterial cultures. Genomic DNA purification. The production of cDNA libraries. Restriction endonuclease and ligases: general characteristics and use in the recombinant DNA technology. Purification of DNA fragments from agarose matrices. PCR-based site-directed mutagenesis: principles and examples. DNA sequencing: the "Sanger's" method and automated sequencing. Analysis of nucleic acids by hybridisation techniques: principles and applications. The expression of recombinant proteins in heterologous systems: an overview. Systems for the inducible expression of recombinant proteins in E. coli. The baculovirus/insect cell system. In vitro coupled transcription-translation.
Working with proteins: the golden rules. Cellular lysis by osmotic shock, mechanical stress and use of detergents: principles, limits and relevance of the methods. Cellular lysis by ultrasonication: principles and applications. Centrifugation of biological samples: instrumentation. Cell fractionation by differential centrifugation. Quantification of proteins in a sample. Protein electrophoresis: principles and instrumentation. The electrophoretic separation of proteins by SDS-PAGE. Protein analysis by isoelectric focusing (IEF): principles and applications. 2D Electrophoresis. "Total" or "group-specific" staining methods to visualise proteins upon gel electrophoresis. Protein detection by immunoblot: western blot and dot-blot.
Immunological tests: general principles. ELISA, RIA and competitive assays. The lateral flow immunochromatographic assay: examples of disposable devices for rapid testing.
Introduction to Liquid Chromatography techniques (LC) for protein purification. Affinity chromatography (AC): matrices modified with group-specific and mono-specific ligands. AC approaches for the isolation of "tagged" recombinant proteins. Ion exchange chromatography (IEC). Hydrophobic interaction chromatography (HIC). Size exclusion chromatography (SEC) and its application to the analysis of protein-protein interactions and protein oligomeric state in solution. Methods to concentrate proteins in a sample.
Study of macromolecular interactions. Pull-down and co-immunopurification. The two-hybrid yeast system (2HYS) for confirming protein interactions and as an interaction cloning approach. The phenomenon of surface plasmon resonance (SPR) applied to quantitative, in real-time study of interactions between macromolecular partners and of protein/small molecule complexes. The in vitro and in vivo analysis of macromolecular complexes dynamics based on the FRET phenomenon: principles and applications.

Upon successful completion of the course, the students will have acquired knowledge and understanding of the taught subjects, and gained skills to autonomously deal with and solve new and/or complex problems on the studied topics. In details, they will know and understand a) the methods, the techniques and the experimental protocols for the production and purification of proteins and nucleic acids to be used in biotechnology and diagnostic field and b) the methods, techniques and experimental protocols for the manipulation and the biochemical/functional characterisation of proteins, nucleic acids and macromolecular complexes. The students will be able to apply this knowledge a) to set up an experimental protocol for the expression and / or purification of proteins; b) to adapt an already published procedure to a specific case, in order to perform a biochemical and/or functional analysis of a given biological macromolecule; c) to develop new functional assays, by properly choosing the experimental methods/reagents/equipments for conducting the experiments. Finally, the students are expected to build up the capability to query the correct sources and databases, and to extract the significant information, developing the learning skills necessary to undertake further study on the subjects covered by the course, with a high degree of autonomy. The students will be able to use the appropriate terminology to communicate the results of "problem solving" activities. Overall, these capabilities will serve as the theoretical basis i) for an informed understanding of other disciplines that the students will encounter in the following of their curriculum, and ii) to perform lab activities aimed at collecting/analysing biochemical and cell biology data.