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 fields. The lessons will treat subjects and problems related to the microbial production of primary and secondary metabolites and the production of recombinant proteins in different prokaryotic and eukaryotic host systems. The classical and molecular genetics methods for the study of functions and gene interactions will be also discussed
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 e Kluyveromyces lactis.
-Baculovirus- insect cell expression systems.
-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