Robust Engineering of Single-Copy Number, Dynamic Genetic Circuits

“具有自动检测和消除自然环境（肠道微生物组，根际）中侵入性病原体（细菌或真菌）的能力的微生物是对抗感染的益生菌的有希望的候选者。挑战是设计一种益生菌微生物，具有高选择系数杀死病原体超过细菌，以及低生长负担，以提高其在社区中的适应性。在这里，我们提出了一种合成生物学方法，用于益生菌工程，以提供抗菌肽（AMP）等治疗药物。目前的许多方法缺乏对潜在遗传电路如何影响微生物在其自然环境中的适应性和殖民行为的全面理解。使用我们实验室开发的成熟的“母机”型微流体装置和自擦除条形码系统的高级版本，我们提出基于振荡器基因电路研究不同的AMP生产策略。这种方法允许使用延时显微镜以单细胞分辨率并行探测数百种不同的设计和数千次重复。此外，通过单细胞分析可以推断基因回路对细胞生长和能量负荷的影响。该项目的初始阶段将包括在微流控装置中的共培养实验中测试不同微流控生产的AMP的功效和效力。这决定了振荡函数下的振幅或面积是否是决定性参数，以及振幅或面积如何根据AMP的作用模式而变化。脉冲式蛋白质生产减少生长负担的假设将通过比较发酵产生的GFP（Potvin-Trottier et al. 2016）和组成型表达来测试。该项目的目的是将整个系统整合到常见益生菌如E. coli Nissle 1917.这允许更精确的计算建模，并且对于释放到其中不存在维持质粒的选择压力的自然系统中是必要的。此外，在这些过程中学到的设计原理将通过将类似的振荡器集成到革兰氏阳性模式生物枯草芽孢杆菌中进行测试。这些生物设计原则旨在定义未来在生物治疗和农业等不同环境中的抗菌和抗真菌应用。"

"Microbes with the ability to automatically detect and eliminate invasive pathogens (bacterial or fungal) in natural settings (gut microbiome, root rhizosphere) are promising candidates for probiotics in combating infections. The challenge is to design a probiotic microbe that has a high selection coefficient for killing pathogens over commensals, as well as low growth-burden to improve its fitness in a community.Here we propose a synthetic biology approach towards the engineering of probiotics to deliver therapeutics like antimicrobial peptides (AMP). Many current approaches lack a comprehensive understanding of how the underlying genetic circuitry is affecting the microbes fitness and colonisation behaviour in its natural setting. Using an advanced version of the well-established "mother machine"-type microfluidic device and a self-erasing barcoding system, both developed in our lab, we propose investigating different AMP production strategies based on oscillator gene circuits. This approach allows to probe hundreds of different designs with thousands of replicates at a single-cell resolution for many generations in parallel using time-lapse microscopy. Furthermore, single-cell analysis allows inference of the gene circuit's effect on cell growth and energetic burden.The initial stages of the project will include testing the efficacy and potency of different oscillatory-produced AMPs in co-culture experiments in the microfluidic device. This determines whether the amplitude or the area underneath an oscillating function is the decisive parameter and how this changes based on the AMP's mode of action. The hypothesis that pulsatile protein production reduces growth-burden will be tested by comparison of oscillatory-produced GFP (Potvin-Trottier et al. 2016) and constitutive expression.The project is then aimed at integrating the entire system into the chromosome of common probiotics like E. coli Nissle 1917. This allows for more precise computational modelling and is necessary for release into natural systems in which no selective pressure for maintaining plasmids is present. Furthermore, the design principles learned during these processes will be tested by integrating a similar oscillator into the gram-positive model organism Bacillus subtilis. These biological design principles are set to define future antibacterial and antifungal applications in diverse settings such as biotherapeutics and agriculture."