There is great interest in engineering organisms with novel or improved function to add to the human gut microbial community; examples include engineering probiotics for increased beneficial function (butyrate production, antimicrobial compounds), or organisms with ability to sense pathogen presence and elicit a targeted response. In vitro fermentation models are increasingly utilized to enable systematic study of the gut microbiome using human fecal samples to inform animal or human studies. However, due to the complexity of the gut microbiome, characterizing engineered organism behavior within a fecal-derived microbial community can be challenging. Use of a mock community would reduce complexity and allow the study of engineered organism persistence and function within more tailored microbial community dynamics. Furthermore, evidence is emerging that more simplified microbial communities can facilitate understanding of microbial competition dynamics that translate to bacterial metabolism behavior in more complex communities. Here, literature surveys, modeling and experimental results are being jointly employed to develop a suitable mock community (anticipating a target of 5–10 organisms) for evaluation of engineered bacteria in increasingly stringent competitive growth environments. A mock community design approach has been created based on carbohydrate metabolism as an indicator of inter-species competition and specific selection criteria. Preliminary mock community studies to investigate probiotic bacteria Lactobacillus spp and E. coli Nissle, engineered to produce plasmid-encoded Green Fluorescent Protein (GFP) in response to an inducer. Each were examined in pairwise co-culture with the gut commensal B. thetaiotaomicron under synergistic growth parameters, representing the lowest level of community competition. Plasmid-engineered E. coli Nissle grown in co-culture showed no interaction with the gut commensal as shown by qPCR. The GFP-encoding plasmid was maintained during co-culture, and GFP fluorescence was demonstrated, although function was negatively impacted by the absence of antibiotic, which is used for plasmid maintenance, indicating a potential change in metabolic state. The systematic complex community analyses of engineered organisms will facilitate feedback for re-design, if necessary, and provide function and persistence validation prior to testing within a more highly complex community environment.

Assembly of mock microbial community for testing engineered microorganisms persistence and function

256th National Meeting (2018)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.