|teoria Metodologie di Genetica||4||I semestre||Antonella Furini, Diana Bellin|
|teoria Metodologie di Microbiologia||1||I semestre||Sandra Torriani|
|laboratorio Metodologie di Genetica||5||I semestre||Diana Bellin, Antonella Furini|
|laboratorio Metodologie di Microbiologia [laboratorio 1° turno]||2||I semestre||Sandra Torriani|
|laboratorio Metodologie di Microbiologia [laboratorio 2° turno]||2||I semestre||Giacomo Zapparoli|
The final aim of the module Methods in Genetics is to provide students the knowledge about the recombinant DNA technology and the methods used for genomic and molecular genetics analyses. Lectures will offer an overview about most common and more innovative genetic methodologies applied for the analysis of genes and their function. The acquired knowledge will allow students to successfully apply routine molecular techniques widely used in genetic studies and to understand and deal also with more innovative methods. Finally this knowledge will allow students to understand the experimental part of scientific papers in genetics.
The module Methods in Microbiology aims to provide students with some tools of knowledge in the sectors of microbiology and biotechnology to enable them to deal with the higher level courses of biotechnology; in particular it helps to understand the potential applications of microorganisms in the agri-food field and the interaction among microorganisms, food, intestinal tract and health.
The course describes basic methods used in genetic analysis and/or molecular biology for DNA and RNA handling. Program includes the following topics:
- Description of animal and plant cell cultures;
- Animal and plant cells genetic transformation;
- Vectors and selection markers, most common cloning techniques, GATEWAY and TOPOCLONING and GOLDENBRAID
(1CFU Prof. Furini);
- Genomic libraries and cDNA libraries: plasmid vectors, Bacteriophage λ, Cosmids, BAC and YAC;
- Transformation of bacterial and yeast cells;
- Use of reporter genes (GFP, YFP) for cellular localization;
- Protoplast preparation and transfection;
- DNA-protein interaction in the gene expression regulation: Chromatin immunoprecipitation (ChIP), Shift assay, luciferase assay
(1CFU Prof. Zenoni)
- Methods for genomic DNA extraction;
- Genome analysis by Southern blotting;
- Molecular markers based on hybridization or PCR and linkage analysis;
- Approaches for gene mapping and genome analysis;
- Sanger DNA sequencing and NGS sequencing;
- Random and site specific mutagenesis, genome editing, CRISPR/Cas9;
- Forward and reverse genetics to study gene function;
- RNA extraction and gene expression analysis through Northern blotting, RT-PCR and Real Time RT-PCR (2CFU Prof. Bellin)
1. Gene cloning in plasmid vector:
Cloning strategy, primer design and PCR reaction preparation, PCR amplicon purification, DNA quantification, TOPOCloniong. E. coli Competent cell transformation. Confirmation of construct, LR recombination, Plasmid DNA purification and verification of the construct (Prof. Furini).
2. Tobacco genetic transformation:
Tobacco plants maintained in vitro culture in sterile conditions. Expant preparation and co-cultivation with agrobacterium. Analysis of construct used for plant transformation. Espant cultivation on selective medium. Analysis of transgenic plants by enzymatic assay (histochemical assay), and by molecular analysis (PCR on genomic DNA) (Prof. Furini).
3. Protoplast transfection for protein subcellular localization:
Cell wall enzymatic digestion for protoplast preaparation. Count and evaluation of the vitality of the protoplasts. Protoplast transfection with vectors carrying cellular markers and microscopic analysis of fluorescent proteins in different cell compartments (Endoplasmic reticulum, vacuole and plasma membrane). (Prof. Zenoni).
4. Marker analysis: use of SSLP and SSR markers and applications examples.
Genomic DNA extraction from model plant Arabidopsis thaliana or from other species using two different extraction methods. Comparison of the performance for the different extraction methods concerning yield and quality based on spectrophotometric analysis and evaluation on agarose gel. Set up and optimization of SSLP and SSR marker analysis on known and unknown genotypes and gel electrophoresis. Sample preparation for SSR analysis on capillary electrophoresis sequencer and SSR analysis. Scoring of results: example for parental analysis, varietal identification and genomic analysis based on linkage (Prof. Bellin).
5. Site-specific mutagenesis to confirm the putative involvement of an aminoacid in the catalytic activity of a protein
Sequence analysis and determination of the site to be mutagenized, primer design for mutagenesis. PCR amplification with designed primers and phosphorylation, ligation and digestion with DpnI for mutagenized plasmid enrichment. Transformation of competent cells. Recovery of transformed colonies and minipreps. Screening of mutants by restriction analysis. Sequence analysis of selected plasmids and discussion on possible applications (Prof. Bellin)
6. Expression analysis of a transgene in a transgenic overexpressor organism by real-time RT-PCR
RNA extraction from transgenic organism and wild type. Evaluation of quality and yield of extracted RNA, DNAse treatment, retrotranscription, and real-time RT-PCR analysis. Evaluation of expression levels for the transgene in wild type and overexpressor by deltadelta Ct method (Prof. Zenoni).
7. Analysis of DNA-protein interaction and evaluation of the binding of a transcription factor to the promoter of a target gene by chromatin immunoprecipitation.
Fixation of two plant tissues characterized by a different expression of a transcription factor. Chromatin extraction from the two tissues, sonication and evaluation by electrophoretic run of DNA fragmentation, immunoprecipitation with a specific antibody against the transcription factor, release of the immunoprecipitated DNA and PCR analysis to evaluate the presence of the target gene in the two tissues analyzed (Prof. Zenoni).
The course program for the Methods in Microbiology module includes the following topics:
- The risk in the laboratory of microbiology. Classification and hazard of biological agents. Genetically modified microorganisms (GMMO). Classes of GMMO application. Biosecurity standards.
- The microbiota of human gastro-intestinal tract. Factors that influence the composition of the microbiota (age, antibiotics, diet, disease). Probiotics and prebiotics.
- The antibiotic-resistant (AR) bacteria in food: potential risks to consumers. The QPS concept. Mechanisms of AR. Transfer of AR genes. New approaches to the study of AR (Prof. Torriani)
There is 1 CFU of exercises during which traditional and biomolecular approaches will apply to:
- the study of bacterial cultures used in the production of functional foods (eg. probiotic fermented milks).
- the detection and characterization of commensal bacteria with antibiotic resistance in products with complex microbiota (Profs Torriani and Zapparoli)
At the end of the course students will present reports about the laboratory experiences both for Methods in Genetics and Methods in Microbiology. The students will do a written examination about all theory and laboratory program covered. The exam will contain both open questions and true/false quiz and exercises for the laboratory part.