GENETIC METHODS FOR BIOTECHNOLOGY
Learning outcomes of the course unit
This course is aimed to highlight the main molecular-genetics methods with regard to their applications both in basic research and in industrial and biomedical area. Potentialities and problematic relative to their use will be focused and discussed. In the first part of the course the potential of microrganisms as cell factories for the production of goods and services will be described. The different approaches for screening, selection and genetic manipulation of industrial strains are described. General arrangement and sectors of application of products of industrial fermentations are 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.
A basic knowledge of genetics, microbiology and recombinant DNA technology is required.
Course contents summary
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).
-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 (E. coli, B. subtilis). 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.
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 interactions.
-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. To address some topics scientific articles will be analyzed and discussed. The teaching materials projected will be made available, nevertheless it is strongly recommended the use of texts for the deepening and study
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.