In Vitro Fermentation Test Bed for Evaluation of Engineered Sense and Respond Probiotics in Polymicrobial Communities

There is great interest in using synthetic biology to engineer organisms with novel or improved function to add to the human gut microbial community; including engineering probiotics for increased beneficial function. The design-test-build-learn approach is commonly used in synthetic biology, largely for circuit design. Engineered bacterial cells are developed and tested in monoculture, under optimal growth conditions that don’t reflect the highly complex environments of the intended application. In contrast to monoculture, characterizing engineered organism behavior within a highly complex microbial community can be challenging. In vitro fermentation models using simplified microbial communities address this challenge by reducing complexity and allowing the study of organism persistence and function within more tailored microbial community dynamics. With increased experimental capacity, in vitro models allow rapid evaluation of multiple parameters to generate knowledge that could then inform design of more physiologically relevant in vivo studies. Here, simplified polymicrobial communities of gut organisms were utilized to investigate engineered probiotic bacteria Escherichia coli Nissle and Lactobacillus plantarum. A simplified community with increasing complexity influenced reporter output with multiple plasmid constructs that encode for varying fluorophores. The function of the engineered E. coli Nissle constructs, designed to produce GFP, was dependent on community composition. L. plantarum engineered to produce GFP showed comparable functional effects. The simplified community of gut bacteria was chosen based on not only abundance on substrate utilization and metabolism. Engineered organism function and persistence tested in the presence of simplified communities of increasing complexity to determine: 1) engineered organism persistence and; 2) effect of engineering on organism persistence; and 3) engineered organism effect on the commensal composition. Testing engineered probiotics using an in vitro fermentation test bed approach can provide critical knowledge for circuit design feedback and functional validation prior to higher fidelity testing within animals or humans.