I am looking for a thesis student (spring 2019 for 6 months) that is interested in metabolic engineering of secondary metabolites by CRISPR, RNAi, and/or overexpression of target genes. Background with biotechnology, and/or molecular biology and cloning is essential. You will clone, transform plants, analyse transgene expression and measure metabolites.

If you are interested, please send a short CV with motivation to julian.verdonk@wur.nl

Petunia scent and colour: Manipulate scent and/or colour in Petunia.

• Investigate the biosynthetic pathway and regulatory elements of scent and colour production. Manipulate gene expression through overexpression, silencing or CRISPR-cas9
• For CRISPR-Cas9 approaches, we will test the constructs in transient expression systems like Nicotiana benthamiana, Tobacco or Petunia, as well as stable transformation.
• Inducible and tissue specific promoters will have to be selected, and part of the project could be the selection of a good candidate promoter

Type of work:

• Tissue culture:
• Plant transformation: Stable, transient, protoplast transformation
• Molecular biology: DNA, RNA, RT-(q)PCR, Cloning (gateway, golden gate, traditional RE cloning)
• Metabolite analysis: HPLC, GC-MS
• Enzyme assays
• Cell biology: subcellular localization of compounds (Anthocyanins located in the vacuole, Carotenoids in the chromoplasts)

Marigold regulation of Carotenoid biosynthesis:

• Manipulate biosynthesis pathway in Marigold to change composition of carotenoids. Change colour or health benefits (Vitamin A). Manipulate gene expression through overexpression, silencing or CRISPR-cas9
• Golden Marigold. Introduce the golden rice construct to see the effects on marigold flowers.
• Study regulatory network of carotenoid biosynthesis. Investigate transcription factors that regulate colour and study the effect of their manipulation on secondary metabolism (colour, scent, health related compounds).

Type of work:

• Tissue culture:
• Plant transformation: Stable, transient, protoplast transformation
• Molecular biology: DNA, RNA, RT-(q)PCR, Cloning (gateway, golden gate, traditional RE cloning)
• Metabolite analysis: HPLC, GC-MS
• Enzyme assays
• Cell biology: subcellular localization of compounds (carotenoids loc. in the chromoplasts)

CRISPR-Cas9 projects

• We have multiple projects to design and test CRISPR-Cas9 approaches in several species. It is the goal to test the constructs in transient expression systems like Nicotiana benthamiana, Tobacco or Petunia.
• Part of this project is the characterization of the target gene. For example, expression during ripening/development, or growth conditions, using qPCR. It is also necessary to clone the target DNA in a binary vector.
• Co-transformation of a construct that contains the target gene or region of the species of interest and the CRISPR-Cas9 construct that will cut it, will give us an idea if it will work.
• This thesis will be a great way to learn the newest molecular biology tools. We will use Golden Gate, Gateway, and traditional restriction enzyme cloning. Also, qRT-PCR, and a lot of bioinformatics will be part of the thesis. Transient agrobacterium transformations, and the start of tissue culture to make real transgenic plants will all be part of your work.

Type of work:

• Tissue culture:
• Plant transformation: Stable, transient, protoplast transformation
• Molecular biology: DNA, RNA, RT-(q)PCR, Cloning (gateway, golden gate, traditional RE cloning)

If you are interested in any of these projects, please send a short CV with motivation to julian.verdonk@wur.nl