GENETIC METHODS FOR BIOTECHNOLOGY
Learning outcomes of the course unit
KNOWLEDGE AND UNDERSTANDING ABILITY.
The course aims to implement the knowledge and skills related to the main genetic methods for the production of recombinant proteins, such as drugs, vaccines and biomolecules of industrial interest, in prokaryotic and eukaryotic microorganisms, in insect cells and in animal cells. During the course the student will also learn the classical and molecular genetic methodologies for the study of gene functions and interactions.
CAPACITY TO APPLY KNOWLEDGE AND UNDERSTANDING
At the end of the course the student should be able to design and deal with the recombinant production of proteins in the different expression systems. It should also be able to design simple experiments to study the functions of essential and non-essential genes and their interactions in the yeast model system.
AUTONOMY OF JUDGMENT.
The student will be able to process what he has learned and recognize situations and problems in which the genetic methods described can be used.
The student will acquire the ability to express himself with appropriate scientific language in the description of the methodologies treated and in the discussion of experimental results. The student's ability to express himself in a clear and.
The student will be able to deal independently with subsequent studies that require knowledge of genetic and molecular methodologies, and will be able to apply the acquired study method to other areas.
A basic knowledge of genetics, molecular biology and recombinant DNA technology is required.
Course contents summary
The course aims to present the main methodologies and the most appropriate experimental approach for the large-scale production of recombinant proteins by exploiting different biological systems for the expression of the cloned gene; from bacterial cells to animal cells. The main applications of biotechnological compounds such as hormones, cytokines, enzymes with therapeutic properties will be discussed. To address the recombinant production of vaccines and monoclonal antibodies, we will recall the fundamental notions of the mechanisms underlying the pathogenicity of bacteria and viruses and the genetic strategies that allow the diversification of immune responses to antigens.
The classical and molecular genetic methodologies for the study of gene functions and interactions in the "yeast model system" will also be presented.
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.
Assessment methods and criteria
The learning assessment will be done through a traditional oral exam which will consist of 3 questions. 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).