Dynamic analysis of microbial genetic adaptation to engineering using microfluidic probing of metabolism status – DYNAMETAFLUID

The Dynametafluid Project aims at developing a microfluidic device for real time and clonal evaluation of metabolic and energetic balance shifts in engineered microbes within controllable and adjustable microenvironments. Each microorganism is encapsulated into a growth medium droplet that acts as an individual micro-bioreactor. For osmotic reasons, evolution of the size of the droplets is directly related to cell energetics. Hence, growth of a single cell into a population and its metabolism are tracked at the same time. Subpopulations of growing cells will be differentially submitted to metabolic perturbations resulting either from light or temperature-induced recombinant gene expression, changes in oxygen or carbon dioxide concentrations, or chemical stresses and their physiological response dynamically analyzed. Based on real time windowed multi-parametric analysis, individual droplets will be selected and recovered to allow complementary genomic and transcriptomic analyses down to single cell level. Genotype, metabolic and transcriptional status of pseudo-clones will be compared in high throughput conditions allowing the establishement of genotype-phenotype relationships at the level of subpopulations of variable sizes. Following a careful validation phase of the hardware involving simple models of recombinant microorganisms (yeast and E. coli), microbial models more representative of metabolic engineering of industrial interest will be designed and analyzed. Among basic questions, consequences of stochastic events affecting single or multistep metabolic engineering directed by episomal vectors will be addressed in relation to the type of vectors and mode of metabolic coupling (intra- or inter-cells) under positive, negative and triggered selections. A focus will be performed on different types of short term (copy number, transcriptional) and long term (genetic drift) adaptive mechanisms that will be characterized both at phenotype, genotype and transcriptome levels. In the last phase of the project, the validated technology will be transferred onto the site of biologist partners for out-of-project use in basic and industrial projects. The project involves three internationally recognized complementary partners specialized in microfluidic, omics and genetic engineering respectively and takes place in close vicinity and interaction with the industry oriented TWB transfer structure.