GENETIC METHODS FOR BIOTECHNOLOGY
Learning outcomes of the course unit
The topics covered during the course aim to provide students with the tools necessary to understand the role of microorganisms in different fields of study and biotechnology application. The principles of large-scale fermentation technologies and examples of microbial biotechnological production of industrial interest will be discussed. Students will also learn, using recombinant DNA technology, to deal with issues related to the production of recombinant proteins in prokaryotes and eukaryotes microrganisms, in insect cells and in animal cells.
A basic knowledge of genetics, microbiology and recombinant DNA technology is required.
Course contents summary
The course consists in the presentation and discussion of the main molecular-genetics methods with regard to their applications both in basic research and in industrial and biomedical area. In the first part of the course the potential of microrganisms as cell factories for the production of goods and services will be described: particular attention will be paid to the different methodological approaches aimed at the selection and genetic improvement of industrial cultivation. Examples of production and use of the main products of industrial fermentations will be considered. In the second part of the course the lessons will treat subjects and problems related to the production of recombinant proteins in different prokaryotic and eukaryotic host systems. Several commercial biotechnology products such as hormones, cytokines, enzymes with therapeutic properties, vaccines and monoclonal antibodies will be taken into account. Finally the classical and molecular genetics methods for the study of gene functions and interactions will be addressed.
MICRORGANISMS AS "CELL FACTORY".
-Microrganisms with biotechnological applications.
- Screening and genetic improvement of industrial strains (bacteria and fungi) by genetic manipulations.
-Amino acids and antibiotics: from the isolation of the microorganism producer to industrial production.
-Microbial biomass production: baker’s yeast and single cell protein (SCP).
- Production of ethanol, citric acid and lactate
-Fermentation technology: raw materials and media composition; batch, fed-batch and continuous process.
GENETIC ENGINEERING AND HETEROLOGOUS PROTEIN PRODUCTION
-Isolation of a gene of interest. Mutagenesis techniques.
THE CHOICE OF THE HOST:
-Analysis and optimization of the gene expression in prokaryotes. Gene expression from strong and regulatable promoters. Fusion proteins: cleavage and use. Increasing protein stability and secretion.
-Analysis and optimization of the gene expression in eukaryotic microrganisms. Yeast expression vectors, constitutive and regulatable promoters. Secretion of heterologous proteins in S. cerevisiae. Other yeast expression systems: Pichia pastoris and Kluyveromyces lactis.
-Baculovirus-insect cells expression system.
-Mammalian cell expression systems. Transient and stable expression. Promoters and reporter genes. Viral vectors. Ex-vivo and in-vivo gene therapy.
THE PRODUCTS OBTAINED WITH THE RECOMBINANT DNA TECHNOLOGY
-Pharmaceuticals and enzymes.
-Monoclonal antibodies as therapeutic agents. Production of antibodies in microrganisms.
STUDY OF GENE EXPRESSION AND FUNCTION.
-Northern Analysis, reporter genes, analysis using arrays.
-Gene interactions: suppressors, synthetic lethal mutations, mutations gene dosage dependent.
-The two-hybrid system.
Donadio e Marino “Biotecnologie microbiche” Casa Editrice ambrosiana, Manzoni M.”Microbioloogia Industriale” Casa Editrice ambrosiana, Glick, Pasternak and Patten “Molecular Biotechnology” ASM press IV edizione, Dale and von Schantz "Dai geni ai genomi" Principi e applicazioni della tecnologia del DNA ricombinante” Edises, Watson J.D et al “DNA ricombinante” Zanichelli. -Teacher's slides and presentations.
The course will be conducted through lectures on specific topics of the program with the aid of power point presentations. The teaching materials projected will be made available to students, nevertheless it is strongly recommended the use of texts for the deepening and study. Some selected topics will be analyzed and discussed with the help of scientific articles.
Assessment methods and criteria
The learning assessment will be done through a final examination. In particular, an oral exam of the traditional type will be carried out with the aim to test the ability of understanding of the various topics and highlight the capacity of the student to make connections between the various topics especially in relation to the advantages and limitations of conventional biotechnology respect to innovative and recombinant biotechnology.