KNOWLEDGE AND UNDERSTANDING
The course aims to teach students the advanced techniques of Molecular Biology used for the isolation of eukaryotic genes and for the study of their expression. During the course, the student will also learn procedures for the expression of exogenous genes in bacteria, unicellular and multicellular eukaryotic organisms.
CAPACITY TO APPLY KNOWLEDGE AND UNDERSTANDING
At the end of the course, students will have acquired the necessary skills to design experimental studies aimed at identifying and isolating a specific gene, to study the modulation of its expression, to express its gene product in a recombinant manner and to generate variants through mutagenesis procedures.
The student will have the basic tools for the analysis of the results obtained from the experimental studies with which to arrive at the understanding of the structure and function of a gene.
During the oral examination, not only the level of understanding of the subject will be tested, but also the ability of the student to express himself in a clear and appropriate scientific language.
Giving the student the ability to understand the experimental studies reported in the most recent scientific works, the course will allow acquiring a certain autonomy both in understanding the most recent technologies used in molecular biology and in designing of new and advanced experimental methods.
Students should have a solid background in Molecular Biology and Recombinant DNA technology
Course contents summary
The course will address the following topics:
- Isolation of a specific gene (cDNA, genome region that contains it).
- Transcriptional analysis of the gene whose cDNA has been isolated.
- Analysis of the structure of the genome region containing the gene of interest.
- Expression in bacteria of the protein coded by the gene.
- Mutagenesis of the gene and of the recombinant protein coded by it.
- Chemical synthesis of DNA and creation of artificial genes.
- Gene Cloning in bacteria other than E. coli.
- Expression of heterologous proteins in B.subtilis.
- Expression of heterologous proteins in Streptomyces.
- Gene cloning in S.cerevisiae and other fungi.
- Expression of heterologous proteins in S.cerevisiae.
- Expression of heterologous proteins in P.pastoris.
- Preparation of expression library in yeast.
- Functional complementation in S.cerevisiae.
- Knock-out gene in yeast.
- Scheme of the two hybrids.
- Gene cloning in animal cells.
- Plasmid vector for transient expression (with and without replicons).
- Plasmid vector for the stable expression (replicons and integration).
- Co-transformation and gene amplification.
- Effect of position and remedies.
- Viral transduction.
- Protein Expression system of baculovirus and vaccinia virus.
- Adenovirus, AAV and retroviruses.
Ingegneria genetica. Principi e tecniche. S.Primrose et al. - Zanichelli
DNA Ricombinante - J.D.Watson et al. – Zanichelli
The course consists of lectures on the main topics covered by the program also with the support of audiovisual media. The teaching material used during the lessons will be provided to the student through the Elly platform
Assessment methods and criteria
The evaluation of the expected learning outcomes is based on an oral test. The ability of the student to design an entire experimental study aimed at solving a hypothetical biomolecular problem will be ascertained.