ADVANCED MOLECULAR BIOTECHNOLOGIES
Academic Year 2023/2024 - Teacher: Vito Nicola DE PINTOExpected Learning Outcomes
Understanding of the technologies used and their purposes.
Course Structure
Lectures and seminar events.
Required Prerequisites
Attendance of Lessons
Sufficient attendance is required, within the framework of the course directions.
Detailed Course Content
Recombinant protein production, a central method in molecular biotechnology.
Protein production in E. coli. Expression of heterologous genes.
Bacterial expression vectors - importance of the promoters used - organisation of the functional elements of the vector.
Mass production of proteins - purification systems - fusion and tagged proteins - bacterial hosts.
Cloning, the fundamental technique for genetic engineering. Advanced cloning methods.
Conventional restriction/ligation cloning. - Cloning with type IIs restriction enzymes. Golden Gate cloning and assembly system. - PCR fragment cloning. TOPO1-TA system.- Ligation-independent cloning (LIC). - Sequence- and ligation-independent cloning (SLIC). - Recombination-based cloning. Gateway system. - Ordered cloning of multiple fragments. - Cloning methods based on PCR reactions. - Mutagenesis based on cloning techniques.
Protein production in eukaryotic cells. - Pro- and cons- of protein production in eukaryotic cells. - Yeasts as protein production systems. - Integration and replication vectors - host cells - culture methods - shuttle vectors. - Expression system in baculoviruses.
Cloning in mammalian cells. - Cloning without a vector. - Primary and secondary cultures. - Expression vectors in eukaryotic cells - nucleic acid transfer methods - liposomes.
Production of therapeutic recombinant proteins in eukaryotic cells in culture - Bioreactors. - Host and vector engineering and maximisation of cell productivity. - Culture methods.
Proteins of biotechnological origin used in therapy.
Discovery, production methods and function of the following classes of proteins. - Therapeutic enzymes - Coagulation factors (Factor VIII and Factor IX) - Thrombolytic agents (tpa, streptokinase- other enzymes. - Hormones and immunomodulators - insulin, glucagon, growth hormone - Interferons and interleukins
The immune response and associated biotechnological molecules.
Humoral and cellular immunity. - Immunoglobulins - poly- and monoclonal antibodies. - Monoclonal antibody production: from the hybridoma method to biomolecular synthesis. Humanised or humanised MAbs - combinatorial libraries - engineered antibody derivatives
Vaccines - systems for selecting and producing the antigens required for immunisation - anti-HIV, anti-influenza vaccines - development and production of anti-Covid 19 vaccines - RNA vaccines
Biotechnological Advanced Therapy Medicinal Products (ATMP) - ex-vivo gene therapies (CAR T Therapy, etc) - viral vectors development and structure and use in gene therapies - in-vivo gene therapies - mutation repair with biotechnological systems.
RNA drugs - protein-coding RNAs - non-coding RNAs: antisense oligonucleotides (ASOs), interfering RNAs, aptamers - mechanisms of action - chemical modifications - SELEX technology - RNA drug transport systems
Patenting and protection of intellectual property: pros and cons - preclinical studies and clinical trials
Protein production in E. coli. Expression of heterologous genes.
Bacterial expression vectors - importance of the promoters used - organisation of the functional elements of the vector.
Mass production of proteins - purification systems - fusion and tagged proteins - bacterial hosts.
Cloning, the fundamental technique for genetic engineering. Advanced cloning methods.
Conventional restriction/ligation cloning. - Cloning with type IIs restriction enzymes. Golden Gate cloning and assembly system. - PCR fragment cloning. TOPO1-TA system.- Ligation-independent cloning (LIC). - Sequence- and ligation-independent cloning (SLIC). - Recombination-based cloning. Gateway system. - Ordered cloning of multiple fragments. - Cloning methods based on PCR reactions. - Mutagenesis based on cloning techniques.
Protein production in eukaryotic cells. - Pro- and cons- of protein production in eukaryotic cells. - Yeasts as protein production systems. - Integration and replication vectors - host cells - culture methods - shuttle vectors. - Expression system in baculoviruses.
Cloning in mammalian cells. - Cloning without a vector. - Primary and secondary cultures. - Expression vectors in eukaryotic cells - nucleic acid transfer methods - liposomes.
Production of therapeutic recombinant proteins in eukaryotic cells in culture - Bioreactors. - Host and vector engineering and maximisation of cell productivity. - Culture methods.
Proteins of biotechnological origin used in therapy.
Discovery, production methods and function of the following classes of proteins. - Therapeutic enzymes - Coagulation factors (Factor VIII and Factor IX) - Thrombolytic agents (tpa, streptokinase- other enzymes. - Hormones and immunomodulators - insulin, glucagon, growth hormone - Interferons and interleukins
The immune response and associated biotechnological molecules.
Humoral and cellular immunity. - Immunoglobulins - poly- and monoclonal antibodies. - Monoclonal antibody production: from the hybridoma method to biomolecular synthesis. Humanised or humanised MAbs - combinatorial libraries - engineered antibody derivatives
Vaccines - systems for selecting and producing the antigens required for immunisation - anti-HIV, anti-influenza vaccines - development and production of anti-Covid 19 vaccines - RNA vaccines
Biotechnological Advanced Therapy Medicinal Products (ATMP) - ex-vivo gene therapies (CAR T Therapy, etc) - viral vectors development and structure and use in gene therapies - in-vivo gene therapies - mutation repair with biotechnological systems.
RNA drugs - protein-coding RNAs - non-coding RNAs: antisense oligonucleotides (ASOs), interfering RNAs, aptamers - mechanisms of action - chemical modifications - SELEX technology - RNA drug transport systems
Patenting and protection of intellectual property: pros and cons - preclinical studies and clinical trials
Textbook Information
Lecture notes. Presentations will be uploaded to Studium. Mongraphical articles or reviews will also be provided to enable students or groups of students to explore individual topics in greater depth.
For basic topics use the text: T:A. Brown, Molecular Biotechnology, 3rd ed. Zanichelli 2022.
For the synthesis of biotechnological drugs we recommend: Vegeto, Maggi, Minghetti, Farmaci Biotecnologici, CEA
There is no single book for the contents of this course, so attendance and study of the bibliographic material provided is essential.
Course Planning
| Subjects | Text References | |
|---|---|---|
| 1 | Presentations by the lecturer and articles of literature will be provided |
Learning Assessment
Learning Assessment Procedures
Oral examination in the form of an interview.
Examples of frequently asked questions and / or exercises
Describe the Gene Editing technology called CRISPR-CAS9.
VERSIONE IN ITALIANO