(640f) Orthogonalized PYR1-Based Biosensors for Multi-Ligand Sensing and Genetic Circuit Construction in E. coli

We have engineered a versatile split-T7 RNA polymerase-based biosensor system in E. coli designed for multi-input, multi-output detection. This sensor utilizes the chemically-induced dimerization of PYR1/ABI1 proteins, which are derived from the abscisic acid signaling pathway in Arabidopsis thaliana. In our design, PYR1 is fused to a C-terminal fragment of T7 RNA polymerase (RNAP), and ABI1 is fused to an N-terminal fragment. This fusion facilitates the reconstitution of functional T7 RNAP in the presence of specific ligands, triggering the transcription of sfGFP. To optimize the system, we regulated C-T7-PYR1 and N-T7-ABI1 using promoters with varied transcriptional strengths. Our focus was on balancing a significant sensor fold change against the toxicity levels of T7 RNAP to the cells. We observed that maintaining a low ratio of N-T7-ABI1 to C-T7-PYR1 optimally achieves this balance. Further, we investigated various mutants of the C-T7 RNAP fragment. These mutants demonstrated orthogonalized transcription of the reporter when matched with specific mutant promoters. Building upon these findings, we tailored our system for simultaneous responsiveness to key organophosphate agrochemicals. Diazinon is a banned pesticide once widely used as a household and agricultural pest control agent. Pirimiphos-methyl is a similarly broad range pesticide used for post-harvest spraying and treatment of structural surfaces. We engineered specific C-T7-PYR1 variants for diazinon and pirimiphos-methyl, aligning them with WT C-T7 RNAP and CTGA C-T7 RNAP, respectively. This approach ensured precise detection and responsive differentiation of these agrochemicals. Our work not only paves the way for the selective expression activation in response to designated compounds but also opens avenues for constructing sophisticated synthetic gene circuits with orthogonalized resource allocation.