Molecular Cell Biology and Biotechnological Applications (2019/2020)
Scientific Disciplinary Sector (SSD)
AGR/07 - AGRICULTURAL GENETICS
The teaching is organized as follows:
Biologia cellulare molecolare teoria
Bioreattori vegetali teoria
Biologia cellulare molecolare laboratorio
Bioreattori vegetali laboratorio
Learning objectives Frontal Lessons -recognize the complexity of the eukaryotic cell and list the sites of production and destination of the proteins with particular emphasis on the plant cell -define the “targeting” and “sorting” concepts; - describe the processes of vesicles formation and fusion and the mechanism of cargo selection; - identify the different paths of the proteins to their final destination; - distinguish the different information necessary for targeting a protein along the default pathway and along the other paths; -follow the post-transductional modifications of proteins along the secretion pathway focusing on quality control processes; - acquire an appropriate scientific language; - apply the acquired knowledge to eukaryotic cell engineering. Laboratory - prepare fresh plant samples for observation with standard and confocal microscopes; -use various types of fluorescence microscopes: conventional, inverted, stereomicroscope, confocal; - choose reporter proteins and fluorescent probes to mark various sub-cellular compartments based on the complementarity of emission spectra and available facilities (types of microscopes and filters); -observe and interpret the intercellular localization of fluorescent reporter proteins in various compartments through fluorescence and confocal microscopy, using specific probes to identify the compartments; - Acquire images under fluorescence and confocal microscopes and analyze them. Bioreactors Module Learning objectives Frontal Lessons -to define the concept of molecular farming; -to distinguish between stable and transient expression of recombinant proteins in plant systems; -to describe the major applications of molecular farming, including the production of nanomaterials, antibodies and vaccines; -to discuss the various strategies to optimise the production of recombinant proteins in plant systems, including the one based on human glyco-engineering, and their fields of application; -to evaluate the impact of different downstream processing strategies in the production system; -to plan production systems for molecules with medical and industrial applications using plants. Laboratory -to design genetic constructs with different fluorescent reporter proteins that accumulate in different sub-cellular compartments; -to prepare constructs with different fluorescent reporter proteins that accumulate in different sub-cellular compartments; -to express transiently those reporter proteins in the leaves of the model host plant species Nicotiana benthamiana; -to measure the yields of the expressed proteins using semi-quantitative western blotting and to compare the efficacy of each single approach.
Cellular Biology Module (Flavia Guzzo)
-Few synthesis sites, many final destinations: protein trafficking in the eukaryotic cell, with particular focus to the compartments and organelles of the plant cell.
- Protein trafficking along the secretion pathway (RE, GA, TGN, PM, V); targeting and sorting.
- The mechanisms of vesicular traffic: formation and fusion of vesicles, their control, cargo selection.
- Protein modifications along the secretion pathway and quality control.
-interpretation of emission spectra of reporter proteins and fluorescent molecular probes for use in fluorescence and confocal microscopy;
-preparation of fresh plant material for observation with fluorescence and confocal microscopes and use of the various type of microscope;
-observation of tobacco leaves transiently expressing fluorescent reporter proteins with various intracellular localizations, prepared in the laboratory module of "vegetable bioreactors" and confirmation of the storage compartments by using appropriate fluorescent probes.
Bioreactors Module (Linda Avesani)
-Introduction to plant molecular farming, state of the art, applications and perspectives;
-Stable and transient expression systems of recombinant proteins in plant systems;
-Nanobiotechnology for the production of nanomaterials in plant systems and their applications;
-Plants in molecular farming: bioreactors and plant systems for direct oral administration;
- Strategies to optimise the production of heterologous proteins in plant systems: from the genetic construct design to sub-cellular targeting;
-The downstream processing in molecular farming;
-The human glyco-engineering for the production of humanized heterologous proteins in plant systems;
-Production in plants of complex molecules: the antibodies;
-Examples of recombinant proteins expressed in plant systems: vaccines and antibodies.
-Design of genetic constructs in silico for the expression of fluorescent reporter proteins in different sub-cellular compartments;
-Realization of the designed genetic constructs;
-Transient transformation using the genetic constructs in N. benthamiana leaves;
-Analysis of the expressed proteins yields.
-to write a thesis on the laboratory experiences
-oral exam on the course subjects.