Fig. 3

Inactivation of the ptTES1 gene using TALEN-based genome editing technique. a Selection of TALEN binding sequences for ptTES1 knockout. A region located in the 4HBT_2 domain of ptTES1 was selected and the 59 nt region consisted of 20 nt for the upstream TALEN (green), a 19 nt spacer where nuclease cleavage may occur, and 20 nt for the downstream TALEN (light blue). Homology regions flanking the Zeocin resistance cassette were designed outside the 59 nt binding region for homologous recombination. b Diagram of ptTES1 gene with targeted disruption by the ShBle cassette. The BleoR resistance cassette (green triangle) was flanked by appropriately 1-kb homology regions (blue boxes) on both sides, and the primer pair ptTES1KO-1/ptTES1KO-2 was used to detect the insertion of the ShBle cassette into the ptTES1 gene. c PCR was used to screen P. tricornutum colonies co-transformed with both TALEN expression plasmid and ShBle knockout plasmid. d Western blotting was used to detect the ptTES1 expression in the ptTES1 knockout mutant and wild-type algal strain. Total soluble proteins were extracted from both strains, separated by 12% SDS-PAGE, blotted, and incubated with the α-ptTES1 antibody. Total soluble proteins were separated and stained with Coomassie brilliant blue as a loading control