Development of Aspirin-Inducible Biosensors in and SimCells.

A simple aspirin-inducible system has been developed and characterized in by employing the promoter and SalR regulation system originally from ADP1. Mutagenesis at the DNA binding domain (DBD) and chemical recognition domain (CRD) of the SalR protein in ADP1 suggests that the effector-free form, SalR, can compete with the effector-bound form, SalR, binding the promoter and repressing gene transcription. The induction of the promoter was compared in two different gene circuit designs: a simple regulation system (SRS) and positive autoregulation (PAR). Both regulatory circuits were induced in a dose-dependent manner in the presence of 0.05 to 10 µM aspirin. Overexpression of SalR in the SRS circuit reduced both baseline leakiness and the strength of the promoter. The PAR circuit forms a positive feedback loop that fine-tunes the level of SalR. A mathematical simulation based on the SalR/SalR competitive binding model not only fit the observed experimental results in SRS and PAR circuits but also predicted the performance of a new gene circuit design for which weak expression of SalR in the SRS circuit should significantly improve induction strength. The experimental result is in good agreement with this prediction, validating the SalR/SalR competitive binding model. The aspirin-inducible systems were also functional in probiotic strain Nissle 1917 and SimCells produced from MC1000 These well-characterized and modularized aspirin-inducible gene circuits would be useful biobricks for synthetic biology. An aspirin-inducible SalR/ regulation system, originally from ADP1, has been designed for strains. SalR is a typical LysR-type transcriptional regulator (LTTR) family protein and activates the promoter in the presence of aspirin or salicylate in the range of 0.05 to 10 µM. The experimental results and mathematical simulations support the competitive binding model of the SalR/ regulation system in which SalR competes with SalR to bind the promoter and affect gene transcription. The competitive binding model successfully predicted that weak SalR expression would significantly improve the inducible strength of the SalR/ regulation system, which is confirmed by the experimental results. This provides an important mechanism model to fine-tune transcriptional regulation of the LTTR family, which is the largest family of transcriptional regulators in the prokaryotic kingdom. In addition, the SalR/ regulation system was also functional in probiotic strain Nissle 1917 and minicell-derived SimCells, which would be a useful biobrick for environmental and medical applications.