Synthetic Auxotrophy as A Pathway for Obligate Commensialism

• Defne Elbeyli, Chemical Engineering, University of Delaware

• Aditya Kunjapur, Chemical and Biomolecular Engineering, University of Delaware

Microbes are being engineered to address issues in both environmental and public health sectors; however, these engineered organisms require a biocontainment strategy to prevent the introduction of hazardous biological material into the surrounding environment. Synthetic auxotrophy is an effective form of biocontainment that relies on recoding organisms to depend on nonstandard amino acids (nsAAs) to maintain essential cellular activities: it has surpassed minimum NIH limits for escapee rates by demonstrating no detectable escape with assay limits between 10-11 and 10-12 escapees per colony forming unit after 100 days of culturing. The deployment of these synthetic auxotrophs outside of a laboratory setting, however, is limited due to the lack of research that surrounds their behavior. Therefore, we aim to create a fully biocontained system where the growth of a synthetic auxotroph is completely dependent on another biocontained strain so the system can be deployed into an open environment without the need for human intervention. To accomplish this goal, we plan to investigate the dynamics of a co-culture that we create between Bacillus subtilis and Escherichia coli synthetic auxotrophs, where the E. coli synthetic auxotroph produces the nsAA needed by B. subtilis. For a successful system, we need to both engineer a synthetic auxotroph to produce our desired nsAA and determine proper conditions for E. coli and B. subtilis to survive a co-culture. We engineer a pre-existing E. Coli synthetic auxotroph for the production of our desired nsAA and establish conditions where it could produce high enough titers in a timeframe that should be favorable to the growth of a dependent strain. Simultaneously, we compare the growth dynamics of both E. coli and B. subtilis and achieve a successful co-culture at various inoculation ratios. Finally, we co-culture our producer E. Coli strain with an existing E. Coli synthetic auxotroph to establish an obligate commensalism in co-culture.