Fig. 2: Switch-testing of bifunctional on- and off-responses.
a Constructs based on T-switch, the 155+165 group, by introducing negative feedback loop control of input signals, namely 147+167 group. lacI gene was co-expressed with sfgfp to stringently repress the promoter activity of PR with the downstream insertion of a LacI-associated operator, LacO. b Fluorescence intensity (FI) monitoring of time course of T-switch circuits by altering temperature from 30 °C to 37 °C right after inoculation to characterize the response-curve under thermal inductions. Cells were grown at 30 °C throughout the pre-cultivation from a single colony. c On- and off-response performance of T-switch constructs shown in a in different growth phases. Recombinant cells were grown for 12 h after changing temperature from 30 °C to 37 °C. Left: distribution and variance of fluorescence, including sfGFP and mRFP, monitored using a flow cytometer; right: quantitative comparisons of on/off performances. Data in b and c are presented as mean ± s.d. of three replicates. d 12 h Time-lapse photography of the temperature-responsive performances of engineered E. coli harboring T-switch, and constructs 155+165, during a shift from 37 °C to 30 °C. Samples comprising 1 µL of the cell suspension were injected and incubated underneath a layer of solid LB with 1.0% agarose (~1.5 mm) containing the relevant antibiotics at 30 °C from overnight cultures at 37 °C. Since the tightness and ultra-sensitivity of tetR family repressor PhlF, the activation of sfGFP lagged behind the quenching of mRFP by at least 4 h.
