Fig. 2
From: Plug-and-play metabolic transducers expand the chemical detection space of cell-free biosensors
Calibration of sensor and output modules for benzoate detection. a BenR binds to the PBen promoter in the presence of benzoate and activates gene expression. Here, BenR is cloned in the pBEAST plasmid (a derivative of pBEST20) and driven by a strong constitutive promoter, OR2-OR1-Pr. The PBen promoter is cloned into another pBEAST backbone and drives expression of superfolder green fluorescent protein (sfGFP). As the system operates without a cellular boundary, multiple plasmids encoding different components of the network can easily be used simultaneously. Plasmid concentrations can then be fine tuned to identify optimal operating conditions. b Optimization of the BenR sensor and reporter modules. Cell-free reactions of 20 µl containing different concentrations of the BenR and reporter plasmids were prepared and their response to different concentrations of benzoic acid were monitored. The white square represents the optimal condition (100 nM reporter and 30 nM BenR plasmid) with the highest relative fluorescence (see Supplementary Fig. 2 and Supplementary Table 1). Reactions were run in sealed 384-well plates in a plate reader at 37 °C for at least 8 h. The heat maps represent the signal intensity after 4 h. Data are the mean of three experiments performed on three different days and all fluorescence values are expressed in Relative Expression Units (REU) compared with 100 pM of a strong, constitutive sfGFP-producing plasmid. See Methods for more details. c Upper panel: The BenR sensor can detect benzoic acid over three orders of magnitude and at concentrations as low as 1 µM. Shaded area around curves corresponds to ±SEM of the three experiments. Lower panel: GFP expression in response to the same range of concentrations of benzoic acid as in the upper panel is easily detectable by eye on a UV table. Source Data are available in the Source Data File
