Educational offer - University of Padova

Syllabus

Prerequisites:	The course requires basic knowledge of biochemistry, cellular biology, microbiology, genetics, and molecular biology. The student should know the structure and the function of both the eukaryotic and the prokaryotic cell. Moreover, the student should be familiar with the structure, the function, and the replication mechanisms of nucleic acids.
Target skills and knowledge:	In the course module of Applied Microbiology, the students will enhance their own knowledge of general microbiology with fundamental concepts of applied microbiology such as advanced systems for the expression and purification of recombinant proteins in eukaryotic organisms, the study of protein-protein interactions, the use of microorganisms as vectors for DNA or protein/peptides delivery for therapeutic/vaccine purposes, etc. Furthermore, the students will be initiated to the practical use of microorganisms for bioremediation and for the production of transgenic plants. In the course module of Genetic Engineering, the students will learn the principles of the recombinant DNA technology, and in particular the methods for cloning and gene manipulation, DNA sequencing, recombinant protein production in procaryotic expression systems will be emphasized. In the practical Laboratory, the students will learn to clone a gene of interest in a plasmid and to use the two-hybrid system for the study of protein-protein interactions in eukaryotic cells.
Examination methods:	The exam will consist in a written test.
Assessment criteria:	Accuracy and completeness of test answers. Appropriateness of language.
Course unit contents:	Lectures Module of Genetic Engineering: Escherichia coli and its natural hosts: plasmids and bacteriophages, mechanisms of conjugation, infection, transformation (natural and artificial), antibiotic resistance. Control of the copy number in plasmid vectors. Recombinant DNA technology: manipulation of purified DNA, restriction enzymes, DNA and RNA polymerases, terminal modification of DNA using kinases and phosphatases. Cloning strategies: DNA ligase, use of linkers and adapters, cloning of PCR products. Prokaryotes cloning vectors: selection of recombinant clones through inactivation of marker genes, construction and use of polylinkers, single stranded M13 vectors, cloning in insertion or substitution lambda vectors. Overview of high-capacity vectors for cloning of genomic DNA (cosmids, fasmids, artificial chromosomes). How to identify and express a cloned gene: synthesis of labeled probes, selection of clones of interest within a cDNA or genomic DNA library, manual and automated sequencing of DNA with the Sanger method, use of universal primers, expression vectors, purification of recombinant proteins in E. coli. Module of Applied Microbiology: Eukaryotic cloning vectors for Fungi (including yeasts), plants and superior animal organisms. Expression and production of recombinant proteins in eukaryotic microorganisms, plants and superior eukaryotic cell culture systems (including the baculovirus expression system) or animals; inducible gene expression systems. Two-hybrid system and related techniques (i.e., one-hybrid system and three hybrid system) to study protein-protein and protein-nucleic acid interactions. Phage display technology. Bacteria, viruses and microbial proteins as vectors to delivery therapeutic genes, DNA vaccines or immunogenic proteins/peptides. Molecular techniques to detect and identify microorganisms or microbial contaminants in biological, environmental, and food samples. Use of microorganisms for bioremediation and in agriculture (production of transgenic plants). Laboratory The practical lessons aim at providing the students with theoretical and practical basis of a number of technologies exploiting microorganisms or their products for biotechnological purposes. The experimental tasks will focus on: 1. Cloning of a gene of interest in a plasmid vector. The molecular cloning of the genes which encode for the proteins that will be then tested for protein-protein interaction by using the Two-Hybrid System, will be performed in suitable plasmid vectors. 2. Use of the Yeast Two-Hybrid System technology to study protein-protein interactions. Students will inoculate and grow yeast cultures, and will prepare selective plates for yeasts. Then, yeast cells will be transformed with the plasmid constructs previously generated. Finally, filter- and liquid-based beta-galactosidase assays will be performed on the transformed yeasts. To this end, protein yeast extracts will be prepared and quantified by using the Lowry method. Results will be analyzed and discussed.
Planned learning activities and teaching methods:	Lectures and practical lessons
Additional notes about suggested reading:	Slides of the lessons and bibliographic material provided by teachers.
Textbooks (and optional supplementary readings)	Glazer AN, Nikaido H., Microbial Biotechnology. --: Cambridge University Press, --. Dale JW, von Schantz M., Plant N., Dai geni ai genomi – Principi e applicazioni della tecnologia del DNA ricombinante. --: Edises, 2013. III Edizione Brown TA, Biotecnologie molecolari. --: Zanichelli, 2007. Reece RJ, Analisi dei geni e genomi. --: Edises, 2006. Primrose S, Twyman R, Old B, Ingegneria Genetica. --: Zanichelli, 2004. Glick BR, Pasternack JJ, Biotecnologia molecolare. --: Zanichelli, 1999. Kun LY, Microbial Biotechnology. --: World Scientific Publishing, --.