Expanding the molecular toolbox in diatoms: developing a transformation system, CRISPR-Cas and Inverse Yeast-1-hybrid

• Main Author: Hopes, Amanda
• Format: Thesis
• Language: English
• Published: 2017
• Subjects: Golden Gate; Sea ice
• Online Access: https://ueaeprints.uea.ac.uk/id/eprint/66542/; https://ueaeprints.uea.ac.uk/id/eprint/66542/1/Final_thesis_corrections.pdf

Diatoms are single celled microalgae with intricately patterned silica cell walls. This cosmopolitan group is a dominant primary producer with many species playing key roles in marine, estuarine and freshwater habitats. Furthermore, due to their silica frustule, lipid production and a range of other chemical and physiological adaptations, diatoms have high potential for biotechnology. Despite their diversity and ecological relevance, molecular tools for diatoms are often underrepresented and limited to a small number of species. This PhD expands the molecular toolbox for two key species: Thalassiosira pseudonana, a model, centric, temperate diatom with a heavily silicified frustule and Fragilariopsis cylindrus, a key, pennate diatom in marine psychrophilic waters and sea-ice. A transformation system has been developed in F. cylindrus leading to the expression of egfp and shble transgenes under the control of an endogenous FCP promoter. This method has been applied to understanding the role of the SITMyb gene, a potential transcription factor with links to silica metabolism, by overexpression. In-silico and in-vitro modelling of the SITMyb gene has been performed and preliminary development of an inverse yeast-1-hybrid system, to elucidate potential transcription factor binding sites, has been carried out. F. cylindrus is the first genetically tractable polar microalgae and appears to be the first psychrophilic eukaryote to be transformed. CRISPR-Cas is a targeted genome editing tool, fast becoming an essential method in any molecular toolbox. This thesis demonstrates development in T. pseudonana by successfully editing the urease gene through a programmed deletion using two sgRNAs. As a model diatom, several molecular tools are already available for T. pseudonana, however this is the first time a targeted knock-out has been achieved in this species. In addition Golden-Gate cloning has been used to produce the construct, giving this method a large degree of flexibility and future potential for multiplexing.