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. It should also be able to address issues related to the production of recombinant proteins.
In particular, the student should be able to:
- Apply the principles of large-scale fermentation technologies
- Apply the most appropriate biotechnological strategies to obtain microbial strains for the production, by fermentation, of primary and secondary metabolites and biomass highlighting the genetic and genetic engineering interventions aimed at improving the production process.
- Define and describe the main molecular methodologies and the most suitable experimental approach for the production of recombinant proteins in prokaryotic and eukaryotic microorganisms, insect and 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 microorganisms 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 addition, technological aspects such as the bioreactors management, the design of fermentation processes and the scaling up from laboratory to industrial production will be introduced.
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, and enzymes with therapeutic properties, vaccines and monoclonal antibodies will be taken into account. Finally, a focus on a specific topic concerning the use of microorganisms as a model system or to study the gene functions and interaction will be proposed.
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
-Microbial biomass production: baker’s yeast and single cell protein (SCP).
- Production of ethanol, citric acid and lactic acid
-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.
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
-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. Selected topics will be analyzed and discussed with the help of scientific articles. Some integrative activities are planned, such as a visit to a company to deepen the subject related to the technology of industrial fermentation.
Assessment methods and criteria
The learning assessment will be done through a traditional oral exam which will consist of 3 questions; one related to fermentation technologies and to the main products of traditional fermentations, one related to prokaryotic and eukaryotic expression systems and one concerning examples of production and applications of recombinant proteins. In the assignment of the final grade will be taken into account the level of knowledge of the different topics (insufficient, superficial, complete and in-depth), the student's ability to apply the theoretical concepts and formulate links between the various topics (sufficient, good, excellent) and of mastery of expression (deficient, simple, clear and correct).