Development of an open source platform for synthetic genomics

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The creation of synthetic genomes has made considerable progress during the past 15 years. In addition to advancing fundamental knowledge in molecular microbiology, bacteria controlled by designer genomes promise to play a very important role in helping society address major challenges of the 21st century. Some of the most obvious applications of engineered microbes include bioremediation, nanofabrication as well as disease treatment. However, only a few significantly modified genome architectures have been explored, mainly because of the relatively high complexity of most model organisms, which significantly increase the cost and workload for this type of project. As a consequence, our understanding of design rules for synthetic genomes remains limited. My laboratory is using the near-minimal bacterium Mesoplasma florum as a fast-growing, non-pathogenic cell model for fundamental research and cell engineering efforts. M. florum is one of the simplest free-living organisms with a genome of only ~800kb encoding fewer than 700 genes. Genetic tools, such as plasmids, antibiotic resistance markers, and efficient transformation protocols have been developed by my research group. In addition, we have cloned the entire M. florum genome as an artificial chromosome in yeast, where it can be genetically manipulated prior to whole-genome transplantation in a recipient bacterium. A deep characterization of the physical and physiological properties of M. florum, a functional annotation based almost exclusively on experimental data as well as a robust in silico whole-cell model that faithfully predicts many phenotypes for this bacterium were also produced by my team. The long-term objective of my research program is to understand the rules that shape genome architecture in bacteria, which we will leverage for the production of synthetic organisms. During the next five years, our main goal is to develop this organism as a freely available open-source genome prototyping platform for the scientific community, and to initiate the investigation of selected synthetic genome variants. More precisely, we propose to generate and characterize a catalog of standardized DNA parts that will be useful to accurately control gene expression in M. florum. We are also interested in developing a new modular and straightforward strategy for large-scale bacterial genome engineering. This novel approach takes advantage of serine recombinases to considerably facilitate the creation of engineered genomes by replacing 40kb chromosome sections by variants assembled using synthetic DNA. We will work on two different designs, a minimal M. florum genome as well as a refactored strain in which genes are clustered in functional modules. Taken together these efforts will help address questions about genome design and cell constraints that would otherwise be very complex and difficult to address with common model organisms, and will contribute to the development of synthetic genomics.