Advances in Phaeodactylum tricornutum nuclear engineering
Abstract
The marine diatom Phaeodactylum tricornutum has the potential to become an excellent platform for the sustainable production of valuable compounds and pharmaceuticals, but currently large-scale engineering of this organism remains a challenge due factors like inefficient genetic transformation protocols and a lack of accurate genomic data. This thesis addresses these two bottlenecks by (i) optimizing an electroporation protocol to P. tricornutum and (ii) remapping genomic data from a scaffolded genome assembly to a telomere-to-telomere genome assembly. An optimized transformation protocol was developed that could consistently transform blunt-ended and DNA with overhangs and yielded up to 1000+ colony forming units per transformation. The method of transgene integration has also been determined to be random integration via non-homologous end joining. Furthermore, the genome coordinates have been updated for 56,624 out of 69,070 annotated genome features to determine their location on the most accurate genome assembly currently available for this organism. In conclusion, the advances made here will streamline genetic engineering for this organism and enables large scale nuclear genome engineering efforts.