Designing and Engineering Gamma-Butyrolactone Signalling for Synthetic Biology

γ-丁内酯是作为“细菌激素”的信号分子，并通过充分研究的信号系统调节抗生素的产生。该项目将利用这些天然系统开发一个多功能的工具包工程丁内酯电路更广泛的应用在合成生物学。设计并开发多种丁内酯生产用的E.大肠杆菌菌株（“信号装置”）。[途径修饰和组装，LS-MS分析]我们将修改信号的化学性质及其产生的动力学。丁内酯化学的多样性将通过即插即用来扩展和设计，以使用先前建立的敏感生物测定来创建新的化学。大肠杆菌菌株（“接收器装置”）。[基于CRISPR的蛋白质工程]将对受体和生物合成酶对进行表征和突变，以产生具有广泛响应特征的受体变体。描述信令和接收设备之间的信令交互。[计算建模]将使用液体共培养和平板生物测定来获得数据，以参数化γ-丁内酯信号传导动力学的现有整体模型，从而预测集成电路的行为。基于该建模，信令和接收器设备的选定组合将被组合在单个E.大肠杆菌菌株，以创建具有不同预测行为的集成电路（开关，生长阶段特定的拨动开关）。我们将进一步应用这些设计的“信号装置”来唤醒沉睡的抗生素基因簇。混合培养E.将培养来自工业合作伙伴的大肠杆菌信号装置和放线菌天然受体菌株，并分析其诱导抗微生物化合物的产生。该项目提供了全面的跨学科培训的方法开创了监督团队，在生物学，生物系统工程，分析和生物信息学的接口，以及在奥德赛治疗，这都是下一代合成生物学家必不可少的工业环境中的培训范围内的关键学科。

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.