Plant molecular methods (2016/2017)



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 sem. Barbara Molesini
laboratorio 2 II sem. Barbara Molesini

Lesson timetable

II sem.
Activity Day Time Type Place Note
teoria Monday 8:30 AM - 10:30 AM lesson Lecture Hall H  
teoria Wednesday 9:30 AM - 11:30 AM lesson Lecture Hall F from Mar 1, 2017  to Apr 19, 2017
teoria Wednesday 9:30 AM - 11:30 AM lesson Lecture Hall F from May 3, 2017  to Jun 9, 2017
laboratorio Monday 1:30 PM - 7:30 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 aimed at developing of transgenic, cisgenic, and intragenic plants. In addition, genome editing tools will be covered.
Particular attention will be paid to the application of these strategy for qualitative and quantitative crop improvement.

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

Syllabus

Molecular mechanism of stable transformation via Agrobacterium: Chromatin targeting of T-complex, proposed model of T-DNA integration; 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); analysis of putative promoter sequence for the presence of cis-acting elements and Chromatin immunoprecipitation assay for identifying the in vivo association of transcription factors with regulatory elements; 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
1) In silico analysis of a target promoter for the identification of possible TF binding sites, chromatin immunoprecipitation for the analysis of a target TF.
2) Design of guide RNA using free bioinformatic software, in vitro transcription of guide RNAs for targeted genome editing and screening of the most effective guide RNAs for the cleavage in vitro of their targets in the presence of Cas9.

Assessment methods and criteria

The exam consists in a written individual exam featuring open-ended questions and exercises.

STUDENT MODULE EVALUATION - 2016/2017