A simple aspirin-inducible system has been developed and characterised in E. coli by employing the Psal promoter and SalR regulation system originally from Acinetobacter baylyi ADP1. Mutagenesis at the DNA binding domain (DBD) and chemical recognition domain (CRD) of the SalR protein in A. baylyi ADP1 suggests that the effector free form SalRr could compete with the effector bound form SalRa, binding the Psal promoter and repress gene transcription. The induction of the Psal promoter was compared in two different gene-circuit designs: simple regulation system (SRS) and positive autoregulation (PAR). Both regulatory circuits were induced in a dose-dependent manner in the presence of aspirin in the range of 0.05-10 μM. Over-expression of SalR in the SRS circuit reduced both baseline leakiness and the strength of Psal promoter. The PAR circuit forms a positive feedback loop that fine-tunes the level of SalR. A mathematical simulation based on SalRr/SalRa 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 the induction strength. The experimental result is in a good agreement with this prediction, validating the SalRr/SalRa competitive binding model. The aspirin-inducible systems were also functional in probiotic strain E.coli Nissle 1917 (EcN) and SimCells produced from E. coli MC1000 ΔminD. These well-characterised and modularised aspirin-inducible gene circuits would be useful biobricks to synthetic bology.
