METACASPASES are cysteine-proteases present in plants, fungi and protozoa. In our group we have identified that METACASPASES can cleave a family of signalling peptides called PROPEPs in a calcium-dependent stresses like wounding . However, several questions remain to be answered like “What are the other substrates of METACASPASES?”. Which stresses can induce METACASPASE activity and how are METACASPASE´s responses modulated? What are the consequences for the plant of their substrate processing? We aim to study this topics and indagate together with the student in this areas of research.
- GATEWAY and GREENGATE cloning: genes amplification (promoter, CDS), genetically encoded sensors, transformation in Arabidopsis and Nicotiana. Generation of CRISPR-Cas9 lines (KO and KD mutants).
- Plant genetic analysis: phenotyping, plant selection, DNA extraction and purification, PCR genotyping.
- Plant expression analysis: GUS staining, RNA extraction, cDNA synthesis, RT-qPCR, protein extraction, SDS-PAGE and Western Blot.
- Recombinant protein production: Escherichia coli BL21 production of proteases and biosensors.
- Confocal microscopy: transcriptional and translational lines and biosensors.
- Protein-protein interaction assays: CoIP, rBiFC, TnT assays.
Period: Practices for >3 months from September 2021 to July 2022.
The student will be involved in the development of innovative molecular tools (transcriptional and translational reporter lines, tagged lines, fluorescently labelled proteins and biosensors). Additionally, the student will validate substrate processing and certify bona fide interactor using both in vivo and in vitro techniques to confirm the results. During the thesis, the student will have the opportunity to generate a hypothesis through all the scientific steps towards hypothesis confirmation. The techniques include different cloning methods, analysis at transcriptome and protein level, gain of expertise in protein-protein approaches, recombinant protein expression and purification, and generation of transgenic and CRISPR lines.