Bacteriophages (phages) drive the evolution of bacteria by antagonistic coevolution and by promoting transfer of genetic material between bacteria. Phages are intimately involved in a range of biological processes from carbon cycling to the maintenance of human health. Despite their global importance the coevolution between natural phage communities and their bacterial hosts remains poorly understood. This project seeks to understand the mechanism behind a Phage-Induced High-Cell-Density (PIHCD) phenotype using a droplet-based microfluidic device termed the Evolution Machine, developed at the proposed institution (ESPCI Paris). Work Package 1 (WP1) will use the Evolution Machine to determine and characterize the phage(s) responsible for the PIHCD phenotype through coinfection with their Pseudomonas fluorescens host. WP2 will determine the mechanistic basis of the PIHCD phenotype using transcriptomics and genetic manipulation. Finally, in WP3 the long term coevolution between phage(s) and host will be investigated using the droplet-level selection capabilities of the Evolution Machine combined with comparative genomic analysis. Through this fellowship ESPCI Paris will gain from my expertise in molecular biology and bioinformatics while I will develop new skills in microbiology (phage-host coevolution and genetic manipulation) and microfluidic technology (Evolution Machine).

Loyal to its tradition in education and training on an interdisciplinary level between physics, chemistry and biology, ESPCI proposes a brand new international doctoral programme – UPtoPARIS. PhD fellows will have the opportunity to explore a unique environment comprising an elite engineer school, 9 joint research units and a new incubator on campus hosting 12 technology-driven start-ups – a recipe for a cutting-edge doctoral training in the heart of Paris. UPtoPARIS is designed to propose a combination of research activities and innovative and competitive supervising, mentoring and career planning. With PhD projects strongly connected to the transferability of basic science, the programme aims to shape new profiles of forward-looking young researchers by giving them knowledge and skills directly linked to the needs of the European and international industry and market. The intersectoral exposure, provided by secondments, workshops and specific guidance, will help young researchers to gain a precious experience in terms of technological development and application through consciously selected topics of European interest, inspired by the KETs, identified by the EU. The 5 Education and Research Chairs of ESPCI (Total, Michelin, Saint-Gobain, Axa and Safran) are also a guarantee for the privileged long-term partnerships with the industry. The 30 incoming excellent doctoral candidates will also reinforce the internationally renowned profile of ESPCI and the region, by increasing the number of foreign PhD students and, consequently, the quality and the diversity of the scientific exchange. The UPtoParis programme will be a pilot project in terms of better recruitment and career guidance procedures with an important impact on the development of high-quality human resources not only on a local level, but also in the European Research Area.

We propose a novel high-throughput method to study the chemo-mechanical coupling in molecular motors using helicases, enzymes that couple ATP hydrolysis to unwinding of the double helix, as a model system. The method combines a cell-free expression system, droplet microfluidics and fluorescent assays with next generation sequencing to test large (up to millions of elements) libraries of helicase mutants for both unwinding and ATPase activity. The aim is to identify all sequence positions that, if mutated, lead to uncoupling, i.e. abolish unwinding while preserving ATPase activity. Once these positions are identified, their distribution on the enzyme sequence and structure will be analysed to obtain insight into the coupling mechanism. Notably the methodology could be applied to systems where the classical, structure-based analysis of the chemo-mechanical coupling is not possible. We will demonstrate our method on the RecQ helicase from e. coli using existing information on this system as a means of validation. Any further insight will be a demonstration of the power of our approach. When fully developed, our method will serve as a discovery tool for the interdisciplinary community working on helicases that includes medical, biological and physical sciences. It may be applied to aspects of helicase biophysics beyond the chemo-mechanical coupling, to other enzyme families and to screen for helicase inhibitors with higher throughput and lower cost when compared to robotic mictotitre-plate techniques. The proposed work will be carried out at the Griffiths Lab (ESPCI Paris) with a secondment at the Crick Institute (London). This project integrates the training in biophysics of the Experienced Researcher with the expertise of the Host Institution on high-throughput methods and the experience of the Secondment Institution on helicases. The Experienced Researcher will be trained in next generation sequencing and fluorescence-based approaches to molecular motors.