Designing and Engineering Gamma-Butyrolactone Signalling for Synthetic Biology

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Gamma-butyrolactones are signalling molecules acting as "bacterial hormones" and regulating the production of antibiotics through well-studied signalling systems. This project will use these natural systems to develop a versatile toolkit of engineered butyrolactone circuits for broader application in synthetic biology. Design and engineer diverse butyrolactone producing E. coli strains ("signalling devices"). [Pathway modification and assembly, LS-MS analytics] We will modify both the chemical nature of the signals and the kinetics of their production. The diversity of the butyrolactone chemistry will be expanded and engineered through plug-and-play to create novel chemistry using sensitive bioassays established previously.Design and engineer diverse butyrolactone-responsive E. coli strains ("receiver devices"). [CRISPR-based protein engineering] Receptor and biosynthesis enzymes pairs will be characterised and mutated to create receiver variants with a broad range of response characteristics. Characterise the signalling interaction between signalling and receiver devices. [computational modelling] Liquid co-culture and plate bioassays will be used to obtain data to parameterise an existing ensemble model of the kinetics of gamma-butryolactone signalling to predict the behaviour of integrated circuits. Based on this modelling, selected combinations of signalling and receiver devices will be combined in a single E. coli strain to create integrated circuits with a spectrum of different predicted behaviours (bistable switch, growth-phase specific toggle switch). We will further apply these designer "signalling devices" to awaken sleeping antibiotic gene clusters. Mixed cultures of E. coli signalling devices and an Actinomyces native receiver strain from the industrial partner will be grown and analysed for induction of antimicrobial compound production. This project provides comprehensive interdisciplinary training in methods pioneered by the supervisory team, across a range of key disciplines at the interface of biology, biosystems engineering, analytics, and bioinformatics, as well as training in an industrial setting at Odyssey Therapeutics, which are all essential for the next generation of synthetic biologists.