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，通过经过充分研究的信号系统调节抗生素的产生。该项目将利用这些自然系统开发一个多功能的工程丁内酯电路工具包，以广泛应用于合成生物学。设计和制造多种生产丁内酯的大肠杆菌菌株（“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和它们产生的动力学。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法创建新的化学。设计和制造多种对丁内酯敏感的大肠杆菌菌株（“接收装置”）。[基于crispr的蛋白质工程]受体和生物合成酶对将被表征和突变，以创建具有广泛响应特征的受体变体。表征信号和接收设备之间的信号相互作用。[计算建模]液体共培养和平板生物测定将用于获取数据，以参数化γ -丁内酯信号传导动力学的现有集成模型，以预测集成电路的行为。基于该模型，选择的信号和接收设备组合将在单个大肠杆菌菌株中组合，以创建具有不同预测行为谱（双稳态开关，生长阶段特定拨动开关）的集成电路。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。大肠杆菌信号装置和来自工业合作伙伴的放线菌原生受体菌株的混合培养将被培养和分析，以诱导抗菌化合物的生产。该项目提供全面的跨学科培训方法，由监督团队首创，涵盖生物学，生物系统工程，分析学和生物信息学的一系列关键学科，以及奥德赛治疗公司的工业环境培训，这些都是下一代合成生物学家所必需的。

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.