Robust Engineering of Single-Copy Number, Dynamic Genetic Circuits

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"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."