Characterisation and development of aspirin inducible biosensors inE. coliNissle 1917 and SimCells

Abstract

AbstractA simple aspirin-inducible system has been developed by employing the Psalpromoter and SalR regulation system originally fromAcinetobacter baylyiADP1, which has been cloned intoE. colifor 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 inA. baylyiADP1 suggests that inactive SalRican compete with activated SalRa, occupying the binding position of Psalpromoter. The induction of the Psalpromoter was compared in two different designs inE. 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 Psalpromoter. A weak SalR expression significantly improve the inducible strength, which is in a good agreement of the proposed hypothesis of SalRi/SalRacompetitive binding. The PAR system provides a feedback loop that fine-tunes the level of SalR, displaying inducible strength. A mathematical model based on SalRi/SalRacompetitive 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 strainE.coliNissle 1917 (EcN) and SimCells produced fromE. coliMC1000 ΔminD. The well-characterised and modularised aspirin-inducible gene circuits would be useful biobricks for bacterial therapy in environment and medical applications.