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 the biorestoration of cultural heritage (altered frescoes 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
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: Expression and production of recombinant proteins in eukaryotic microorganisms(including the baculovirus expression system), 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. 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 the biorestoration of cultural heritage . 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, plasmid DNA extraction, endonuclease restriction and sequencing.Plasmids containing cloned gene will be transfected into eukaryotic cells and expression evaluate. 2. Isolation of microorganisms from water samples, using molecular methods (RNA extraction, reverse transcription, PCR-end point, Real-time PCR) 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, --.