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. 2016 Jul 25;6:30234. doi: 10.1038/srep30234

Figure 4. Results of SSA assay in E. coli cells and detection of mutation in the potato callus.

Figure 4

(A) Structure of the plasmids for SSA assay containing a divided Luc (firefly luciferase) gene and the expected gene that will be generated by the specific recombination on the overlapped regions. “Divided” indicates the structure of the reporter plasmid that contains a fusion gene for RLuc and 2A peptide followed by the divided Luc. The divided Luc gene is inactivated by insertion of the target sequence of TALENs (shown by “target”). Lu- and -uc show parts of divided Luc genes, in which 430-nucleotides region are repeated. “Revertant” indicates the reporter gene, which will be generated after the appropriate TALEN reaction will induce the site-specific gene recombination at the overlapped region. 35S: 35S promoter; RLuc, Renilla luciferase; 2A, self-cleavable 2A peptide; target, the target sequence of TALENs; NosT, NOS terminator. (B) Results of SSA assays. Assays were performed using a set of plasmids, a TALEN plasmid and a reporter plasmid, pSSARLab or pSSARLcd, which are corresponding to “Divided” on the panel A. pSSARLab contains the target sequence by TALENa and TALENb that are encoded in TALENab as a TALEN plasmid. pSSARLcd contains those of TALENc and TALENd in TALENcd. “ab” and “cd” show the names of TALEN plasmids and reporter plasmids used. Dual luciferase assays were performed using the cell extract of E. coli transformed both by the plasmids for the SSA assay and TALENs. Relative values of the Luc activity to the RLuc activity are shown (n = 5). (C) DNA sequences of the regions corresponding to the target site of the TALENs. WT, #38, #14, and #02 show the nucleotide sequences of the wild-type gene and three mutants of the potato callus, respectively. Gaps indicate the region of nucleotide deletion and a site of insertion. An underlined letter shows the inserted nucleotide in #02, and a reversed letter indicates an A to T substitution. Bars show target sites of TALENs, TALENa and TALENb, and HindIII site.