Fig. 4: Electroporation and assembly of two non-overlapping fragments in P. tricornutum.

a The episome pPtGE31_ShBle was amplified as two fragments (F1: fragment 1, F2: fragment 2). The fragments were adjusted to be equimolar before combining them into an assembly mixture (F1 + F2). b A plasmid map of pPtGE31_ShBle and a depiction of the different possible orientations post-assembly. The episome contains algal selection markers for Ntc^R and Zeo^R, a bacterial Cm^R selection marker, as well as a yeast CEN and ARS sequences. F1 and F2 do not share any overlapping sequences and thus can assemble in two different orientations. Primer sets were designed to span the two junctions, A and B, where recombination is expected to occur. The amplicons will vary in size depending on the orientation of the fragments. c Screening three E. coli transformants per algal colony using primers spanning junctions A (top panel, expected size of 238 or 359 bp) and B (bottom panel, expected size of 553 or 432 bp). DNA was isolated from algal transformants that were electroporated with F1 + F2 and selected initially with nourseothricin or d zeocin. Dilute pPtGE31_ ShBle and water were used for the positive and negative controls, respectively. Double asterisks (**) indicate the E. coli colonies that were sent for whole plasmid sequencing.