Electronic Thesis and Dissertation Repository

Thesis Format

Alternative Format

Degree

Master of Science

Program

Biochemistry

Supervisor

Karas, Bogumil J.

2nd Supervisor

Gloor, Gregory B.

Co-Supervisor

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.

Summary for Lay Audience

Our planet is being destroyed in humanity's quest for natural resources, leading to things like deforestation, biodiversity loss, and climate change. To ensure that we can fulfill a demand for natural resources in the future and maintain our civilization, we need to find an alternative and sustainable ways to produce natural resources without causing any more harm to our planet. To address this, researchers have proposed using engineered microorganisms, specifically photosynthetic organisms like marine microalgae, as cell factories to produce compounds of interest. One of the best candidate microbes for this purpose is called Phaeodactylum tricornutum. By engineering this microbe, researchers have been able to use it to produce chemicals involved in creating plastics, pharmaceuticals, and even COVID-19 diagnostic tests. Many genetic tools have been developed for this microbe to make engineering easier, but there is still a lot that needs to be done to improve this microorganism’s potential for industrial use.

My thesis focuses on improving methods to deliver and integrate custom DNA into this organism and also to accurately identify where in the organisms DNA all of its genes and other genetic information lie, as new technology has shown that where researchers previously believed all the genes were located is not actually accurate. I have been able to establish a simple, efficient, and reliable protocol to introduce custom DNA into this organism’s genetic code. I’ve also demonstrated that this method can integrate custom DNA randomly into the organism’s genetic code and can be used to inactivate and investigate the function of the organism’s genes. I’ve also identified the correct location of 56,624 out of 69,070 genes and gene-like features in this organism’s genetic code and have provided potential reasons for each of the remaining 12,446 features as to why finding out where they are actually located within the organism’s DNA is a challenge. Overall, the progress I’ve made in this thesis brings us closer to being able to introduce larger fragments of DNA with more complicated instructions into this organism and boosts its potential for use in creating valuable resources in an environmentally friendly way.

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