Broad-Host-Range Genetic Tools for Observing Microbial Consortia

Abstract

Microbial communities are complex assemblages that are key to ecosystem stability and human health. Synthetic ecology aims to design and construct microbial consortia with reduced complexity that enable innovative applications beyond monocultures. However, their robust and performance-based design is constrained by limited knowledge of growth dynamics and derived binary interactions that are critical for community functioning. Decoupling population dynamics and inferring interspecies interactions from strain-specific fitness data remains a major challenge due to the difficulty of monitoring and quantifying species-specific growth in mixed microbial communities. Existing methods have the disadvantage that they are not suitable for fast, scalable, high throughput applications such as those needed at the interface between synthetic biology and microbial ecology, where the screening of large design spaces associated with the construction and observation of synthetic co-cultures is required. In this thesis, a standardized platform functioning as a tractable tool for the interrogation of population dynamics based on measurements of strain-specific fluorescence in microbial co-cultures was developed. This was accomplished by constructing a set of broad-host-range plasmids in the BASIC environment that constitutively express fluorescent reporter proteins and estimating the ecological fitness of species in terms of specific growth rate (µ) and carrying capacity (K) from static and time-course optical density and fluorescence measurements through regression analysis of bacterial growth models. Experimental investigation of model binary co-cultures constructed from six model and non-traditional bacterial hosts demonstrated successful decoupling of population dynamics and inference of interspecies interactions in 96-well plates based on fluorescence. Furthermore, the results emphasize the need for the right choice of genetic tools for a meaningful interrogation of co-culture dynamics and inference of interactions, consistent with the finding that the suitability of fluorescence as a surrogate for bacterial biomass depends on the combination of host species and fluorescent protein. It is anticipated that this toolkit can contribute to applications in synthetic microbial ecology and biotechnology by providing a flexible, scalable, and reproducible approach to decouple populations dynamics in synthetic co-cultures.