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Full-Text Articles in Biochemistry, Biophysics, and Structural Biology
Advances In Phaeodactylum Tricornutum Nuclear Engineering, Mark Pampuch
Advances In Phaeodactylum Tricornutum Nuclear Engineering, Mark Pampuch
Electronic Thesis and Dissertation Repository
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 …
Practical Applications And Future Directions Of Genetic Code Expansion: Validation Of Novel Akt1 Substrates And The Design Of A Synthetic Auxotroph Strain Of B. Subtilis, Mcshane M. Mckenna
Practical Applications And Future Directions Of Genetic Code Expansion: Validation Of Novel Akt1 Substrates And The Design Of A Synthetic Auxotroph Strain Of B. Subtilis, Mcshane M. Mckenna
Electronic Thesis and Dissertation Repository
In Chapter 1, site-specifically phosphorylated variants of the oncogene Akt1 were made in Escherichia coli using the orthogonal translation system that enable genetic code expansion with phosphoserine. The differentially phosphorylated variants of Akt1 were used to validate newly predicted Akt1 substrates. The predicted target sites of the peptide substrates were synthesized and subjected to in vitro kinase assays to quantify the activity of each Akt1 phosphorylated variant towards the predicted peptide. A previously uncharacterized kinase-substrate interaction between Akt1 and a peptide derived from RAB11 Family Interacting Protein 2 (RAB11FIP2) was validated in vitro. Chapter 2 describes the preliminary development of …