IC OF BIOMOLECULAR AND RICOMBINANT TECHNOLOGIE
Academic year and teacher
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- Versione italiana
- Academic year
- 2020/2021
- Teacher
- GIUSEPPE FORLANI
- Credits
- 12
- Curriculum
- BIOTECNOLOGIE PER L'AMBIENTE
- Didactic period
- Primo Semestre
Training objectives
- This course is aimed at teaching the techniques and the basic methodologies to study and analyze nucleic acids and gene expression.
Course of Molecular and Recombinant Technologies (Dr. Alessia Finotti):
The course is aimed at teaching the techniques and the basic methodologies to study and analyze nucleic acids and gene expression, with special attention to the recombinant DNA technology.
The practical laboratory exercise will give the possibility to the student to apply the acquired knowledge and will focus on the most used methods in the common research laboratories. At the same time students will learn the use of basic instruments (e.g. the use pipettes and other common laboratory instruments), and will be addressed on the experimental steps for the analysis of the expression of a specific gene target: from the RNA extraction, its spectrophotometric quantification and qualitative analysis by electrophoresis on agarose gel; the production of cDNA by reverse transcription and quantification of a specific mRNA by real-time quantitative PCR.
KNOWLEDGE AND UNDERSTANDING
The student will learn:
- correct use of scientific terms
- the techniques used to purify and quantify nucleic acids
- the techniques used for genomic and transcriptomic analyses
- the basic elements to build a recombinant vector
- the different type of recombinant vectors.
ABILITY TO APPLY KNOWLEDGE AND UNDERSTANDING
The student will know:
- the DNA and RNA extraction methods and for its analysis
- the approach for genomics and transcriptomics analysis
- which technique should be used for gene cloning experiments
- the suitable vectors used for recombinant protein production
- the vectors used to analyze and modify gene expression.
Plant Molecular Biology.
The course will provide basic and specific knowledge of the molecular mechanisms that regulate the functioning of the plant organisms, with particular attention to the phenomena that are unique in plants. These skills are described taking into consideration possible modifications and applications in agriculture and industry.
KNOWLEDGE AND UNDERSTANDING
The student will learn:
The structure of plant genomes (differences in size, composition and structure in monocot and dicot genomes)
Function and regulation of gene expression in plants
The methods for the genetic modification of plants (Agrobacterium tumefaciens and direct methods)
Some of the applications of transgenic plants and the related problems
The response mechanisms of higher plants to different types of stress (biotic or abiotic) and possible improvements, with emphasis on plant secondary metabolism
The mechanisms of gene silencing in plants
The basics of plant breeding and molecular breeding
ABILITY TO APPLY KNOWLEDGE AND UNDERSTANDING
The student will know how:
to use the correct terminology of plant molecular biology
to understand how to manipulate the modular structure of plant gene
to design experimental strategies of study applied to crops
to critically evaluate the experimental results. Prerequisites
- Course of Molecular and Recombinant Technologies (Dr. Alessia Finotti):
Knowledge of Chemistry, Biology, Biochemistry and Molecular Biology.
Plant Molecular Biology:
Basic knowledge of Plant Physiology and Molecular Biology. Course programme
- Course of Molecular and Recombinant Technologies (Dr. Alessia Finotti):
The course includes 40 hours (5 CFU) of classroom didactic lecturing and 12 hours (1 CFU) of laboratory exercises.
The lessons raise two issues:
FIRST SECTION: Methodologies for the analysis of nucleic acids and their expression and/or modulation (20 hours):
- Extraction and purification of DNA, quantification and analysis: electrophoresis using agarose and Polyacrylamide gels, capillary electrophoresis;
- Techniques for genomic analysis: the employment of enzymes for DNA manipulation (e.g. restriction enzymes, polymerases, ligase), Southern blotting, Polymerase Chain Reaction and its variant, gene sequencing and Next Generation Sequencing;
- Extraction and purification of RNA and introduction to microRNAs;
-Assays for gene expression studies: reverse transcription, quantitative real-time PCR, digital PCR and gene expression arrays;
SECOND SECTION: Techniques of DNA modification, cloning vectors, expression vectors, transfection systems, and biotechnological applications (20 hours):
- Enzymes for DNA cloning and basic principles of recombinant technologies;
- Molecular cloning vectors and their applications (plasmids, bacteriophages, cosmids, ecc.); genomic libraries production;
- Transfection and other methods of introducing foreign DNA into living cells;
- Coning approaches for gene expression study and gene expression modulation: use of RNA interference and gene silencing;
- Assay for promoter and transcription factors analyses;
-Introduction to gene therapy vectors;
-Methods for recombinant protein production: vectors, expression systems for recombinant proteins, pharming, examples of therapeutic applications;
-Monoclonal antibodies.
The hours for the specific sections are indicative.
Plant Molecular Biology.
Lessons (48 h) are structured in different topics, with increasing complexity, the normal duration is about 2 hours each. The following topics will be examined:
Plant in vitro cultures and micropropagation, role of hormones and applications -- The plant gene, the role of the promoter and the terminator, structural modularity -- Methods for plant genetic transformation, Agrobacterium tumefaciens and direct methods, transformation vectors, working protocols -- Applications and issues associated with the production and growth of transgenic varieties -- Gene expression regulation in plants, antisense RNA and RNA interference, applications -- Organization, structure and characteristics of plant genomes, size, repetitive elements and variability -- Plant breeding in species of agronomic interest, classic and molecular methods for cross and selection – Plant responses to stress and pathogens, new applications – Plant secondary metabolism – Advanced techniques for genetic manipulation of crops. Didactic methods
- Course of Molecular and Recombinant Technologies (Dr. Alessia Finotti):
The course is structured in 40 hours of classroom didactic lecturing.
During didactic lecture the course topics will be explained. In the classroom it’ll be also suggested scientific journals, websites, as references and explainer videos. The powerpoint presentation of the topics will be provided by the teacher after the lesson (PDF format).
Suggested books are recommended for a more simple understanding of the basic concepts, but they do not talk about all the arguments explained by teaching and learning in the classroom.
In addition to the 40 hours of lectures (5CFU), it will be held a laboratory exercise (1CFU) divided in a section of practical laboratory experience and a section of preparation and analysis of the results carried out using PowerPoint lectures, data analysis and exercises with the possible assistance of videos and animations.
For the practical laboratory exercise, the students will be divided into groups, and will work individually or in pairs, on these topics: the extraction of total RNA from biological matrices, its spectrophotometric quantification, qualitative and quantitative analysis of the RNA extracted by gel electrophoresis of agarose, retrotranscription and qPCR real time.
Plant molecular biology
The course is structured in 48 hours of classroom lecturing. During each lecture the topics will be explained in details, using figures and tables taken from scientific journals, websites, and videos. The presentation of the topics will be provided by the teacher after the lesson (MP4 format).
Suggested books are recommended for a more simple understanding of the basic concepts, but they do not talk about all the arguments explained in the classroom. To help the students to assess their level of preparation, at regular intervals 4 lectures are devoted to self-assessment tests or discussion. Learning assessment procedures
- Molecular and Recombinant Technologies (Dr. Alessia Finotti):
The written exam includes 31 questions with multiple choice (with 5 answers only one of which is correct) and will be carried out OMR modules. The exam will last up to 60 minutes.
For DSA students, the test procedure will be evaluated individually.
To pass the exam (vote 18/30) it will be necessary to correctly answer at least 18 questions. The vote of 30/30 and honors will be achieved by answering correctly to 31 questions. No penalty points will be counted for wrong answers or unspecified answers.
Plant Molecular Biology.
Exam by interview: the student will be asked three general questions, followed by more in-depth questions that may wander to other subjects of the course. It is particularly valued the ability to relate the different topics of the course and the ability to critically describe knowledge acquired.
Written exam: it consists of 42 questions (about one for each lecture) requiring short open answers or multiple-choice answers. The exactness and the completeness of each answer is evaluated and scored 1 point (correct), 0.5 point (incomplete or only partially correct), or 0 point (incorrect or missing). A total score of at least 18 is needed to pass the exam.
The final vote of Biomolecular and Recombinant Technologies (IC) is the average of the scores obtained in the two modules: Plant Molecular Biology and Molecular and Recombinant Technologies. Reference texts
- Molecular and Recombinant Technologies (Dr. Alessia Finotti):
Slides were shown during the frontal lectures (PDF format); other material provided by the teacher.
Principal suggested text:
-Biotecnologie Molecolari. Principi e tecniche. Terry A. Brown - ZANICHELLI
Any further information on the following texts:
-Metodologie biochimiche e biomolecolari. M. Maccarrone - ZANICHELLI
-Genomi 4. IV edizione, 2018. Terry A. Brown - EDISES
-DNA ricombinante. Geni e Genomi. J.D. Watson et al. - ZANICHELLI
-Analisi dei Geni e Genomi. Richerd J. Reece - EdiSES
-Compendio di Biotecnologie Farmaceutiche. Maria Luisa Calabrò - EdiSES
-Metodologie Biochimiche. Bonaccorsi di Patti, Contestabile, Di Salvo - ZANICHELLI
Plant Molecular Biology.
Notes and course material used during the lessons.
Buchanan B., Gruissem W., Jones R. Biochimica e Biologia Molecolare delle Piante. Ed. Zanichelli
