- Docente: Paolo Bernardo Trost
- Credits: 7
- SSD: BIO/04
- Language: Italian
- Moduli: Paolo Bernardo Trost (Modulo 1) Francesca Sparla (Modulo 2)
- Teaching Mode: Traditional lectures (Modulo 1) Traditional lectures (Modulo 2)
- Campus: Bologna
- Corso: Second cycle degree programme (LM) in Molecular and cellular biology (cod. 8021)
Learning outcomes
At the end of the course, the student will have in-depth knowledge of modern developments in specific areas of plant physiology and the main methodologies targeting the expression of recombinant proteins in plants and their characterization. In particular, the student is able to: - analyze and discuss topics of genomics, redox biology, physiology of abiotic and biotic stress in higher plants; - critically evaluate the scientific literature on the topics covered; - analyze fragments of DNA and transform bacterial cells; - purify recombinant proteins by chromatographic techniques; - conduct analysis of biochemical and molecular characterization of the protein product.
Course contents
The course consists of Module 1 (lectures, 4 credits, Prof. Trost)
and Module 2 (individual experimental laboratory, 3 credits, Dr.
Sparla).
Module 1 (Prof. Trost)
Arabidopsis thaliana as a model species in plant biology.
Nuclear and plastid genome. Endosymbiotic gene
transfer.
Oxidative stess and redox signaling. Production and
detoxification of ROS in plants. Photosynthesis and
photoinhibition. Mitochondria. NADPH-oxidase. Thiol-based redox
signaling.
Abiotic stress. Water stress: osmotic adjustment and
abscisic acid.
Biotic stress. Classes of plant pathogens and
pathogenesis. Constitutive defenses of plants. Basal immunity and
vertical resistance (guard hypothesis and gene for gene model).
Oxidative burst, nitric oxide and hypersensitive response. Systemic
responses.
Module 2 (Dr. Sparla):
Day 1: extraction and analysis of plasmid DNA by
restriction endonuclesi. Calculations and discussion.
Day 2: transformation of E. coli BL21 (DE3) and
selection of transformants on medium with kanamycin. NBT-staining
to verify the production of ROS in the wound response of A.
thaliana leaves.
Day 3: liquid culture of a colony grown on plate.
Separation in SDS-PAGE and subsequent Western blot analysis of
plant proteins.
Day 4: induction of the expression of the recombinant
protein by IPTG; bacterial growth curve. Incubation with secondary
antibody conjugated to alkaline phosphatase and colorimetric
detection. Calculations and discussion.
Day 5: cell lysis and recombinant protein purification
by metal affinity chromatography; desalting of the protein
sample.
Day 6: denaturing gel electrophoresis of the purified
recombinant protein; measurement of protein concentration.
Calculations and discussion.
Day 7: kinetic analysis of the recombinant enzyme.
Calculations and discussion.
Day 8: absorption spectra of the recombinant protein;
assessment of the nature of the flavin cofactor. Calculations and
conclusions.
Readings/Bibliography
- Rascio et al., Elementi di Fiologia vegetale, Edises, second edition
- Taiz Zeiger, Plant physiology, fifth edition, Sinauer Associates
- Updated reviews on key issues (provided by the teacher)
- Power point files
- Detailed protocol of the laboratory module.
Teaching methods
Module 1: Lectures Module 2 (laboratory): Each student is required to carry out the experimental laboratory activities individually. The teacher will give a theoretical introduction to the lab and continuous technical assistance. The results obtained will be critically discussed.
Assessment methods
At the end of the laboratory Module 2 the students will have to pass a written test of verification of the experimental activity carried out. The students who pass the written test, will have access to the oral examination on the topics of Module 1.
Teaching tools
The course will take place in classrooms with PC projection. All lectures will be given with power point presentations. The files of power point presentations will be made available to students at the end of the course (downloadable files from AMSCAMPUS). The lab course will take place in a didactic laboratory with single workstations, equipped with centrifuges, spectrophotometers and electrophoretic cells.
Office hours
See the website of Paolo Bernardo Trost
See the website of Francesca Sparla