Dual-luciferase assays

The Renilla and firefly-luciferase sequences were obtained from the pTH727-CEN-RLuc/staCFLuc plasmid (http://www.addgene.org) and were concatenated with a linker sequence GGTCGACGGATCCCCGGG between them, for the purpose of keeping correct protein folding. This Renilla-linker-firefly fragment was further inserted into the p426 plasmid and was expressed under TDH3 promoter. The sequences of AAGAAGAAG, AAGAAGAAA, AAGAAAAAG, AAAAAGAAG, AAGAAAAAA, AAAAAGAAA, AAAAAAAAG, and AAAAAAAAA were individually inserted upstream of the coding sequence of firefly luciferase. Individual plasmids were transformed into BY4742. The plasmid-containing yeast clones were selected on SC–uracil plates.

The activity of luciferase was detected as described in a previous study [79]. Briefly, transgenic yeast strains were individually cultivated in the SC–uracil medium (150 μL) at 30 °C overnight. Ten microliters of each yeast culture was transferred into a fresh SC–uracil medium (140 μL) and was cultivated for another 4 h in a 96-well plate. Passive lysis buffer (40 μL, Promega, E1910) was added per well for cell lysis. Twenty-five microliters of the suspension was mixed with 25 μL firefly luciferase substrate (Promega, E1910) and was incubated for 20 min at 25 °C. Firefly luciferase activity was measured by the Synergy multi-mode reader (BioTek). Twenty-five microliters of Stop-and-Glo reagent (Promega, E1910) was added and mixed, which was incubated for another 20 min before the measurement of Renilla-luciferase activity.