Figure 2.

Construction and optimization of the SINGER system in Salmonella VNP20009

(A) Schematic representation of the US-inducible transgene expression switch. In the absence of US, the transcriptional repressor TlpA₃₉ forms a homodimer, binds to its specific promoter P_TlpA, and blocks LuxCDABE expression. Upon US, the mild temperature elevation leads to the dissociation of TlpA₃₉ from a homodimer to a monomer and dissociation from its promoter P_TlpA, which drives LuxCDABE expression.

(B) Quantification of transgene expression by engineered bacteria under various induction temperatures.

(C) Induction-time-dependent transgene expression kinetics at 39°C.

(D–F) Screening various ribosome binding sites (RBSs) (D), the number of tandem repeats of TlpA₃₉ binding element (O_TlpA) (E), and constitutive promoters of differing strengths (P_const) to drive TlpA₃₉ expression (F).

(G) Heatmap visualization of the correlation between the temperature of bacteria solution and the examined irradiating acoustic parameters.

(H) Schematic experimental procedure for SINGER-mediated transgene expression.

(I) Irradiation-time-dependent SINGER-mediated transgene expression kinetics.

(J) SINGER-mediated transgene expression in different bacteria strains.

All samples in (B)–(F) were maintained at 37°C after the indicated period of temperature induction (for a total experimental duration of 12 h from the start of induction to the sample collection) before the bioluminescence signals were quantified using a microplate reader. All samples in (I) and (J) were maintained at 37°C after the indicated period of US irradiation (for a total experimental duration of 12 h from the start of the US stimulation to the sample collection) before the bioluminescence signals were quantified using a microplate reader. Data in (B)–(F), (I), and (J) are presented as means ± SD; n = 3 independent experiments. Each data point represents the mean of three technical replicates.

See also Figure S1 and Tables S1–S3.