Plant molecular methods (2015/2016)



Course code
4S003257
Credits
6
Coordinator
Barbara Molesini
Academic sector
BIO/04 - PLANT PHYSIOLOGY
Language of instruction
Italian
Teaching is organised as follows:
Activity Credits Period Academic staff Timetable
teoria 4 II semestre Barbara Molesini
laboratorio 2 II semestre Barbara Molesini

Lesson timetable

II semestre
Activity Day Time Type Place Note
teoria Monday 8:30 AM - 10:30 AM lesson Lecture Hall H  
teoria Friday 9:30 AM - 11:30 AM lesson Lecture Hall F  
laboratorio Monday 1:30 PM - 6:30 PM laboratorio Laboratory Laboratorio di Biotecnologie genetiche  

Learning outcomes

At the end of the course, the student will acquire the technical skills for the design of various types of genetic constructs aiming at the development of transgenic, cisgenic, and intragenic plants. In addition, genome editing tools will be covered.
Particular attention will be paid to the application of these methods for crop improvement.

Supplied educational material:
Power point lessons, relevant research articles and reviews.

Syllabus

Molecular mechanism of stable transformation via Agrobacterium; transgene integration, stability, methylation and silencing; strategies to avoid transgene silencing (e.g. site-specific recombination for precise and clean transgene integration); promoters used for genetic constructs (constitutive, spatiotemporal, inducible, synthetic); coupling synthetic promoters with synthetic transcription factors for coordinated expression of multiple genes; multigene engineering; reporter genes; artificial miRNA and target mimicry; cisgenesis and intragenesis; cisgenic and intragenic genetic constructs; strategies to remove marker genes from transgenic plants; artificial programmable DNA nucleases (ZFNs and TALENs) and RNA-guided DNA nucleases (Type II CRISPR-Cas9 of Streptococcus pyogenes) for genome engineering; molecular mechanism of CRISPR-Cas9; sgRNA design; minimization of off-target activity (e.g. Cas9 nickase and double nicking strategy and alteration in the length of the sgRNA); application of CRISPR-Cas system beyond genome editing (e.g. CRISPR interference, gene regulation, and cargo delivery); genetic constructs for genome engineering using the CRISPR-Cas9 system, screening of mutants generated by CRISPR system (e.g. RFLP analysis and T7 endonuclease I assay); hybridization between nucleic acids and DNA/RNA labelling.

Lab. experiences:
Histochemical localization of Mtp::GUS expression during Medicago truncatula root development: Composite plants generation using Agrobacterium rhizogenes; analysis of the transgenic state of adventitious roots and GUS staining.

Design of sgRNAs using free bioinformatic softwares, efficient production of sgRNAs and efficacy testing of sgRNAs for the cleavage in vitro of their targets in the presence of Cas9.

Assessment methods and criteria

written exam

Student opinions - 2015/2016