A Modular Receptor Platform To Expand the Sensing Repertoire of Bacteria

Abstract

Engineered bacteria promise to revolutionize diagnostics and therapeutics, yet many applications are precluded by the limited number of detectable signals. Here we present a general framework to engineer synthetic receptors enabling bacterial cells to respond to novel ligands. These receptors are activated via ligand-induced dimerization of a single-domain antibody fused to monomeric DNA-binding domains (split-DBDs). Using E. coli as a model system, we engineer both transmembrane and cytosolic receptors using a VHH for ligand detection and demonstrate the scalability of our platform by using the DBDs of two different transcriptional regulators. We provide a method to optimize receptor behavior by finely tuning protein expression levels and optimizing interdomain linker regions. Finally, we show that these receptors can be connected to downstream synthetic gene circuits for further signal processing. The general nature of the split-DBD principle and the versatility of antibody-based detection should support the deployment of these receptors into various hosts to detect ligands for which no receptor is found in nature.

Figure 1

Engineering synthetic receptors using the split-DBD principle (A) Overview of the LexA-based split-repressor system. LexA DBD was fused to VHH-Caffeine. The monomeric chimeric receptor is expressed in the cytosol upon IPTG induction. In the presence of caffeine, the chimeric receptor dimerizes and binds to the LexA operator, blocking expression of the reporter gene. (B) Response of cells harboring plasmids encoding LexA-VHH-Caffeine and LexA-VHH-Control to increasing concentrations of IPTG and caffeine. (C) Fold repression for the two LexA-VHH fusions in response to increasing concentrations of IPTG and caffeine. For each IPTG concentration, fold changes were calculated from (B) relatively to cells grown without caffeine (lower row).

Figure 2

Response of LexA-VHH-Caffeine to increasing concentrations of caffeine at 25, 50, and 100 μM IPTG induction. Upper panel: flow-cytometry data of LexA-VHH-Caffeine response to caffeine at different expression level. Lower panel: titrations curves of LexA-VHH-Caffeine in response to increasing concentrations of caffeine and at different expression level. Error bars: standard deviation between three independent experiments performed in triplicate. *p < 0.05, and **p < 0.01, compared with signal in the absence of caffeine.

Figure 3

A prokaryotic transmembrane receptor using a single-domain antibody for ligand detection. (A) General architecture of synthetic transmembrane receptor using the split-DBD principle. The DBD and Juxtamembrane of the CadC transcriptional activator were fused to an Leu(16)TM, an external linker, and VHH-Caffeine or VHH-Control as LBD. (B) Principle of transmembrane receptor activation. Genes encoding CadC-VHH fusions are placed under the control of the pLacO1 promoter. The N-terminal CadC DBD is located in the cytosol and the C-terminal VHH in the periplasm. In the presence of caffeine, the chimeric receptor CadC-VHH-Caffeine undergoes ligand-induced dimerization and activates downstream reporter gene expression. (C) Response of CadC-VHH-Caffeine and CadC-VHH-Control to increasing concentrations of caffeine at different expression level. (D) Activation fold of the two CadC-VHH fusions. For each IPTG concentration, fold changes were calculated from (C) as in Figure 1.

Figure 4

Response of CadC-VHH-Caffeine to increasing concentration of caffeine at 25, 50, and 100 μM IPTG induction. Upper panel: flow cytometry data of CadC-VHH-Caffeine response to caffeine at different expression level. Lower panel: titration curves of CadC-VHH-Caffeine response to increasing concentration of caffeine at different expression level. Error bars: standard deviation between three independent experiments performed in triplicate. *p < 0.05, and **p < 0.01, compared with signal in the absence of caffeine.

Figure 5

Optimizing transmembrane receptor signal-to-noise ratio through linker engineering. (A) Schematic diagram of the 12 receptor variants tested. (B) Quantification of the response of 12 receptor variants to 100 μM caffeine upon 50 μM IPTG induction. Error bars: standard deviation between three independent experiments performed in triplicate. *p < 0.05, and **p < 0.01, compared with signal in the absence of caffeine.

Figure 6

Connecting synthetic receptors to downstream genetic circuits. (A) General architecture of LexA-VHH-Caffeine connected to the BetI inverter. LexA-VHH-Caffeine controls BetI expression, which controls the GFP expression. (B) Response of cells containing the LexA-VHH-Caffeine receptor connected to the BetI inverter to increasing concentrations of IPTG and caffeine. (C) Fold activation of the LexA-VHH-Caffeine/BetI inverter circuit.