Teaching code

4S003254

Credits

12

Coordinatore

Antonella Furini

Language

Italian

Scientific Disciplinary Sector (SSD)

AGR/07 - AGRICULTURAL GENETICS

Moodle Seminars

The teaching is organized as follows:

Learning outcomes

The final aim of the Methods in Genetics UL of the Methods in Genetics and Microbiology course is to provide to the 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 molecular genetics.
The module Methods in Microbiology aims to provide students with the 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.

Program

The course program for the Methods in Genetics module includes the following topics:
Theory
- Description of animal and plant cell cultures;
- Animal and plant cells genetic transformation;
- Methods for bacterial transformation;
- Vectors and selection markers, most common cloning techniques, GATEWAY and TOPOCLONING;
- Plasmid and genomic DNA extraction;
- Selection of recombinant colonies;
- Use of reporter genes (GFP, YFP) for cellular localization;
- Protoplast preparation and transfection (Prof. Furini)
- Methods for genomic DNA extraction;
- Genome analysis by Southern blotting;
- Molecular markers based on hybridization or PCR and linkage analysis;
- Alternative approaches for gene mapping and genome analysis;
- Sanger DNA sequencing and introduction to NGS sequencing;
- Random and site specific mutagenesis and introduction to genome editing;
- Application of mutagenesis by forward and reverse genetics to study gene function;
- RNA extraction and gene expression analysis through Northern blotting, RT-PCR and Real Time RT-PCR (Prof. Bellin)

Laboratory
1. Gene cloning in plasmid vector with the insertion of a signal peptide:
Cloning strategy, primer design and PCR reaction preparation. PCR amplicon purification, digestion of PCR product and plasmid vector with restriction enzymes. Elettrophoretic analysis and band excision from gel. Band elution, band DNA quantification. Plasmid dephosphorilation. Vector and fragment ligation. E. coli medium preparation. Competent cell preparation. Plasmid DNA purification and verification of the prepared construct.
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).
3. Protoplast transfection for protein subcellular localization:
Tobacco plants maintained in vitro culture in sterile conditions. Cell wall enzymatic digestion for protoplast preaparation. Protoplast transfection with vectors carrying cellular markers and microscopic analysis of fluorescent proteins in different cell compartments (Endoplasmic reticulum, vacuole and plasma membrane). (Prof. Furini)
4. Mapping of genes involved in function of interest by a mutant based approach.
Genomic DNA extraction from model plant Arabidopsis thaliana 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 marker analysis on parents of an F2 population obtained by outcrossing a selected mutant affected in phenotype, extraction of genomic DNA from progenies of the population, segregation analysis of optimized SSLP on the crossing population by marker scoring on agarose gel, linkage analysis among markers and to identify the genomic location of the mutation and gene involved in the phenotype
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.
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. Bellin)

The course program for the Methods in Microbiology module includes the following topics:
Theory
- 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.
- Microorganisms and food: the wine. The role of yeast and bacteria. Spontaneous and guided fermentations. Selection of starter cultures. New perspectives in microbiological research.

Laboratory
There are 2 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,
- the assessment of the effect of various yeasts, including a genetically modified strain of Saccharomyces cerevisiae, on the fermentation kinetics and production of metabolites of interest for the quality of wine in microvinification trials

Examination Methods

Concerning the Methods in Genetics module at the end of the course students will present two written reports about the laboratory experience. The students will do a written examination about all theory and laboratory program. The final aim of the exam is to test the learning of arguments faced in both the theory and laboratory part of the course. The exam will test all topics included in the program and will contain both open questions and true/false quiz and exercises for the laboratory part. All together the exam includes two parts, to be done together, yielding a total of 90 points (40 points for the Prof. Furini part and 50 points for the Prof. Bellin part). The final points will be translated in a mark out of thirties.

Concerning the Methods in Microbiology part at the end of the module, a report on the practical exercises carried out in the laboratory is scheduled; this report will have a maximum score of two points. The assessment of learning is through a final written exam. The written test will contain six questions for each of which a maximum of five points will be assigned .The test is aimed at assess and verify the skills and knowledge acquired in the issues addressed throughout all the course program. The examination will be considered positive if at least 18 points will be assigned to the student.
Only for students who will pass both module exams, final mark will be the weighted average of the marks.