Figure 6.

Demonstration of the use of HiBiT reporter system in TXTL. (A) eGFP fluorescence measurement of the HiBiT-GFP fusion proteins. Fusion proteins were expressed in TXTL at 30 °C for 8 h, followed by the fluorescence measurement. (B) The schematic of how LgBiT-carrying E. coli cell-free extract works. The plasmid coding LgBiT gene under the sigma 70 promoter is transformed into E. coli Rosetta 2 strain. The cell-free extract is made with that strain; thus, the extract contains LgBiT. Once HiBiT is expressed in TXTL, HiBiT produces luminescence by reconstituting a full luciferase with LgBiT. (C) End-point luminescence assay with the fusion HiBiTs. The fusion HiBiTs were expressed in the LgBiT-containing TXTL at 30 °C for 8 h. After the expression, 1 µM Furimazine was added, followed by luminescence measurement. (D) Luminescence kinetics measurement with the LgBiT-containing TXTL. HiBiT plasmids and Furimazine were added at the TXTL reaction set up and incubated at 30 °C. The luminescence was measured every 5 min during the TXTL reaction. N-GFP, N-terminal eGFP fusion with HiBiT; C-GFP, C-terminal eGFP fusion with HiBiT; Control, reaction without enzyme expression. The graphs show means with error bars that signify SEM (n = 3).