Characterisation and development of aspirin inducible biosensors in E. coli Nissle 1917 and SimCells

A simple aspirin-inducible system has been developed by employing the P sal promoter and SalR regulation system originally from Acinetobacter baylyi ADP1, which has been cloned into E. coli for characterisation of gene circuits and induction of novel SimCells (simple cells). Mutagenesis at the DNA binding domain (DBD) and chemical recognition domain (CRD) of the SalR protein in A. baylyi ADP1 suggests that inactive SalR i can compete with activated SalR a , occupying the binding position of P sal promoter. The induction of the P sal promoter was compared in two different designs in E. coli: simple regulation (SRS) and positive autoregulated system (PAR). Both regulatory systems 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 system reduces both baseline leakiness and inducible strength of P sal promoter. A weak SalR expression significantly improve the inducible strength, which is in a good agreement of the proposed hypothesis of SalR i /SalR a competitive binding. The PAR system provides a feedback loop that fine-tunes the level of SalR, displaying inducible strength. A mathematical model based on SalR i /SalR a competitive binding hypothesis was developed, which not only reproduces the observed experimental results but also predict the performance of a new gene circuit design. The aspirin-inducible systems were also functional in probiotic strain E.coli Nissle 1917 (EcN) and SimCells produced from E. coli MC1000 ΔminD. The well-characterised and modularised aspirin-inducible gene circuits would be useful biobricks for bacterial therapy in environment and medical applications.