Directed Evolution of Synthetic E. coli Predator Population Using Microfluidic Emulsion Droplet Platform

Microbes have evolved strategies to adapt to nutrient limitations and environmental stresses like competition within microbial communities. Because of these interactions, bacteria have accumulated the ability to produce various specialized metabolites under specific environmental conditions. Activation of these cryptic pathways can provide us with insights into how microbial communities interact as well as access to potentially novel bioactive metabolites for industrial and biomedical use. Here, we use emulsion droplets as a culturing and screening platform to mimic the in vivo predator-prey ecosystem by encapsulating single cells from two competing Escherichia coli populations.

As a proof of principle, we engineered a model predator-prey system in E. coli, such that each droplet contains a ‘defective-predator’ and a ‘prey’. A defective-predator has a single nucleotide polymorphism (SNP) to knockout the production of quorum sensing (QS) molecules. An evolved predator activates the engineered cryptic pathway to produce QS molecules required for killing the prey. Therefore, an evolved predator can grow to a higher density compared to a defective-predator within the droplets. By iterating the growth of evolved predators in the emulsion, we could increase its population density to detectable level.

Currently, we have optimized the platform for efficient droplet production, incubation and droplet sorting. The sorting system can detect even a single GFP-labelled E. coli cell (pSB1C3_K608010) encapsulated within a 50µm droplet. This enables us to selectively screen for functional Predator from a mixed population even at very low densities, compared to a suspension culture. Next, this competition based directed evolution strategy will be applied to harness the SNP reversion in a mutagenized defective-Predator population to produce the quorum sensing molecules under defined co-culture conditions. If successful, this technology can be applied to non-model organisms such as Streptomyces to evolve and screen for variants that produce specialized metabolites with bioactive properties.