Learning objectives
KNOWLEDGE AND UNDERSTANDING.
The course aims to implement the knowledge and skills related to the main genetic methods to produce 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.
APPLYING 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.
MAKING JUDGMENTS. The student will be able to apply the acquired knowledge to identify situations and problems where the described genetic methodologies can be effectively utilized. They will develop the ability to critically evaluate experimental data and select the most appropriate experimental approaches to solve specific problems in the field of biotechnology. COMMUNICATION SKILLS. 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. LEARNING SKILLS. 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.
Prerequisites
A basic knowledge of genetics, molecular biology and recombinant DNA technology is required.
Course unit content
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.
Full programme
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
microorganisms.
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.
-New vaccines.
-Monoclonal antibodies as therapeutic agents. Production of antibodies in
microorganisms.
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
Bibliography
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. -Original papers provided by the teacher .
Teaching methods
The course is conducted in person through lectures on specific program topics, using PowerPoint presentations. The slides and other instructional materials used in the lessons, including peer-reviewed scientific videos, will be provided to students through the Elly platform. Course enrollment is required to access these online resources.
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 considered 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).
Other information
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