Plant molecular methods (2018/2019)



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

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laboratorio 2 II semestre Barbara Molesini

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Learning outcomes

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MM: teoria
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At the end of the course, the student will acquire the theoretical and practical basis for the design of various types of genetic constructs aimed at developing transgenic, cisgenic, and intragenic plants. In addition, genome editing tools will be covered. Particular attention will be paid to the application of these strategies for qualitative and quantitative crop improvement.
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MM: laboratorio
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The laboratory practice will offer to the students the ability to design relevant experiments aimed at addressing two biological questions and to critically evaluate experimental data.

Syllabus

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MM: teoria
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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; reporter genes (transcriptional and translational fusion constructs); tools employed for the study of DNA-protein interactions: Chromatin immunoprecipitation assay, DNA electrophoretic mobility shift assay, DNA pull-down assay; 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 transformed plants (e.g. via site-specific recombinase under the control of inducible promoters); 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); applications 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, T7 endonuclease I assay, heteroduplex mobility assay); hybridization between nucleic acids and DNA/RNA labelling. Supplied educational material: Power point lessons, relevant research articles and reviews.
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MM: laboratorio
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- In vitro transcription for dsRNA generation, application of dsRNA in planta after flower emasculation to induce RNA silencing of a repressor of ovary growth. - Design of guide RNA using free bioinformatic software, template preparation by PCR using overlapping primers, in vitro transcription of guide RNAs 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 will ascertain the students’ knowledge on the topics of the lectures and lab practices. The exam, for both attending and non-attending students, will consist in a written individual exam featuring open-ended questions and exercises.


STUDENT MODULE EVALUATION - 2017/2018