Figure 4: Suggested strategies to clone sequences of interest into diatom episomes.

(a) Genes or pathways of interest can be assembled into p0521s replacing the URA3 gene. Incoming DNA should be prepared with sequence overlaps to the plasmid regions flanking URA3 and assembled in yeast spheroplasts with counter-selection on 5FOA. (b) Smaller sequences interest (equivalent in size to single expression cassettes) can be assembled into pPtPuc3 using Gibson assembly or other cloning strategy. Sequences can be introduced anywhere in the plasmid, but we typically insert at the 3′ region of the CEN6-ARSH4-HIS3 sequence. (c) Any existing plasmid can be modified with the yeast CEN6-ARSH4-HIS3 sequence amplified from p0521s, pPtPuc3 or other source to enable episomal replication in P. tricornutum or T. pseudonana. The oriT sequence should also be included (if it is not already present in the vector sequence) if conjugation from E. coli is chosen as the method of plasmid introduction.