Characterisation and development of aspirin inducible biosensors in E. coli and SimCells

A simple aspirin-inducible system has been developed and characterised in E. coli by employing the P sal 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 SalR r could compete with the effector bound form SalR a , binding the P sal promoter and repress gene transcription. The induction of the P sal 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 P sal promoter. The PAR circuit forms a positive feedback loop that fine-tunes the level of SalR. A mathematical simulation based on SalR r /SalR a 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 SalR r /SalR a 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. Importance An aspirin-inducible SalR/ P sal regulation system, originally from Acinetobacter baylyi ADP1, has been designed to E. coli strains. SalR is a typical LysR-type transcriptional regulator (LTTR) family protein and activate the P sal promoter in the presence of aspirin or salicylate in the range of 0.05-10 μM. The experimental results and mathematical simulations support the competitive binding model of SalR/ P sal regulation system, in which the effector free form SalR r competes with effector bound form SalR a to bind P sal promoter and affect gene transcription. The competitive binding model successfully predicted that a weak SalR expression would significantly improve the inducible strength of SalR/ P sal regulation system, which is confirmed by the experimental results. This provides an important mechanism model to fine-tune transcriptional regulation of LTTR family, which is the largest family of transcriptional regulator in the prokaryotic kingdom. In addition, the SalR/ P sal regulation system was also functional in probiotic strain E.coli Nissle 1917 (EcN) and minicells derived SimCells, which would be a useful biobrick for environmental and medical applications.