## 4 Materials and Methods
### Diatom Transformation, Selection, and Subculturing  
The plasmids were verified by next-generation sequencing and were then amplified in an _E. coli_ Epi 300 strain containing the pTA-MOB plasmid to enable conjugation with wild-type diatoms, as described in the literature [38]. _P. tricornutum_ (Culture Collection of Algae and Protozoa, CCAP 1055/1), was kindly provided by Prof. [PERSON]. The transfer of plasmid DNA to _P. tricornutum_ via conjugation from _E. coli_ was performed as described by [PERSON] et al. [38]. For this process, 250 \(\mu\)L of wild-type _P. tricornutum_ culture was adjusted to a density of 10\({}^{8}\) cells/mL, while the _P. tricornutum_ cell density was obtained by plating 1 mL of wild-type _P. tricornutum_ on 1/2 \(\times\) L1 1% agar plates and growing them at 18 \({}^{\circ}\)C under cool fluorescent lights (75 \(\mu\)E m\({}^{-2}\)s\({}^{-1}\)) on a light/dark cycle of 16/8 h for 4 days. Prior to transformation, 1 mL of L1 media was added to each agar plate, and the cells were first scraped and then harvested via pipetting in a sterile tube. The cells were then diluted and mounted in an improved Neubauer hemacytometer (BLAUBRAND(r) counting chamber, MilliporeSigma Canada Ltd., Oakville, ON, Canada) to be counted, then the cell concentration was adjusted to 5.0 \(\times\) 10\({}^{8}\) cells mL\({}^{-1}\). A volume of 50 mL of _E. coli_ culture containing the assembled and pTA-MOB plasmids was grown at 37 \({}^{\circ}\)C under agitation to reach an OD\({}_{600}\) of 0.9, then the volume was centrifuged at 3000\(\times\)\(g\) for 10 min and resuspended in 500 \(\mu\)L of SOC media. Conjugation was initiated by adding 200 \(\mu\)L of _P. tricornutum_ to 200 \(\mu\)L of _E. coli_ cells. The cell mixture was then plated on 1/2 L1, 5% LB, 1% agar plates, incubated at 30 \({}^{\circ}\)C for 90 min in the dark, and then transferred to 18 \({}^{\circ}\)C in the light and grown for 48 h. Two days later, 1 mL of L1 media was added to the plates and the cells were collected by scraping, then a volume of 200 \(\mu\)L of cells was plated on 1/2 L1, 1% agar plates supplemented with zeocin at 50 \(\mu\)g mL\({}^{-1}\) for selection and were incubated at 18 \({}^{\circ}\)C under light (75 \(\mu\)E m\({}^{-2}\)s\({}^{-1}\)). Two weeks later, positive colonies appeared and were streaked again on 1/2 L1, 1% agar plates supplemented with zeocin at 50 \(\mu\)g mL\({}^{-1}\) for the verification of plasmid stability. The recombinant colonies of _P. tricornutum_ were checked via cPCR. Four positive colonies were retained from each construct, from which the plasmids were extracted for further confirmation. The extracted vectors were then amplified in _E. coli_ Epi300; these were extracted using the EZ10 kit and sequenced using next-generation sequencing via Illumina MiSeq technology at the Massachusetts General Hospital's Center of Computational and Integrative Biology (MGH CCIB DNA Core, Boston, MA, USA). In parallel, transformed _P. tricornutum_ culture was launched from a single colony until obtaining an OD\({}_{680}\) nm of 0.06-0.1. The bioengineered _P. tricornutum_ strains were then sub-cultured every 10 days in a 1/3 volume ratio of fresh L1 Si\({}^{-}\) medium containing zeocin (50 mg\(\cdot\)L\({}^{-1}\)).