Figure 2: Demonstration that P. tricornutum episomes replicate as stable, circular, low-copy plasmids (a–e), and expression and localization of proteins encoded on the P. tricornutum episome p0521s (F-I).

(a) Cultures of P. tricornutum containing p0521s (clone 9, Supplementary Fig. 3), were subcultured in seawater medium for 28 days with or without antibiotic selection and plated (Supplementary Fig. 5). DNA from five antibiotic-resistant colonies from each culture (Supplementary Fig. 5D) was recovered in E. coli and isolated plasmids were separated by agarose gel electrophoresis. Shown are rescued plasmids derived from separate P. tricornutum colonies that were initially subcultured for 28 days without (lanes 1–5) or with (lanes 6–10) antibiotic selection. ‘M’ designates supercoiled marker⁴¹ and ‘C’ designates the original plasmid (isolated from clone 9) introduced into P. tricornutum. Arrow denotes supercoiled plasmid band. (b) Stability of p0521-Se containing a 49-kb S. elongatus fragment. Ten independently transformed P. tricornutum lines containing p0521-Se were subcultured in liquid media with selection for 60 days, followed by episome rescue and separation of plasmids by agarose gel electrophoresis. Arrow denotes supercoiled plasmid band. (c) Plasmids extracted from P. tricornutum were untreated, treated with exonuclease, ClaI endonuclease or a combination of exonuclease and ClaI. Treated plasmids were transformed into E. coli and the number of transformed colonies was plotted (error bars indicate one s.d. of the mean from three biological replicates). (d) Agarose gel electrophoresis of plasmids extracted from P. tricornutum and treated with nucleases. Lanes from left to right: (1) 1 kb⁺ ladder (NEB), (2) p0521s control (from E. coli), (3) p0521s exonuclease-treated (extracted from P. tricornutum), (4) p0521s untreated (extracted from P. tricornutum), (5) 1 kb⁺ ladder (NEB), (e) Copy number of p0521s in P. tricornutum determined by qPCR. Cm (Cat gene) and His (HIS3 gene) are loci found on the episome backbone; Ure (urease, protein ID 29702) and NR (nitrate reductase, protein ID 54983) are loci encoded on P. tricornutum nuclear chromosomes 18 and 20, respectively; Rbc (RuBisCO small subunit) and CytB (Cytochromoe B) are loci found on the P. tricornutum chloroplast and mitochondrial chromosomes, respectively. Error bars denote one s.d. of the mean from three biological replicates. (f) Wild-type P. tricornutum, fluorescence measured with GFP settings. (g) P. tricornutum expressing CFP translationally fused to beta-carbonic anhydrase (Protein ID 51305) localized to the chloroplast pyrenoid encoded on plasmid p0521s. (h) P. tricornutum expressing YFP translationally fused to mitochondrial urea transporter (Protein ID 39772) encoded on plasmid p0521s. (i) P. tricornutum expressing GFP localized to the cytoplasm encoded on plasmid p0521s. Scale bar for f–i indicates 5 μm.