Mandell, D. J. | bioRxiv Science

bioRxiv2020

Synthetic auxotrophy remains stable after continuous evolution and in co-culture with mammalian cells

Understanding the evolutionary stability and possible context-dependence of biological containment techniques is critical as engineered microbes are increasingly under consideration for applications beyond biomanufacturing. While batch cultures of synthetic auxotrophic Escherichia coli previously exhibited undetectable escape throughout 14 days of monitoring, the long-term effectiveness of synthetic auxotrophy is unknown. Here, we report automated continuous evolution of a synthetic auxotroph using custom chemostats that supply a decreasing concentration of essential biphenylalanine (BipA). After 100 days of evolution in three separate trials, populations exhibit no observable escape and are capable of normal growth rates at 10-fold lower BipA concentration than the ancestral synthetic auxotroph. Allelic reconstruction of three proteins implicated in small molecule transport reveals their contribution to increased fitness at low BipA concentrations. Mutations do not appear in orthogonal translation machinery nor in synthetic auxotrophic markers. Based on its evolutionary stability, we introduce the progenitor synthetic auxotroph directly to mammalian cell culture. We observe containment of bacteria without detrimental effects on HEK293T cells. Overall, our findings reveal that synthetic auxotrophy is effective on timescales and in contexts that enable diverse applications. One Sentence SummaryTo ascertain whether life inevitably finds a way, we continuously evolve an Escherichia coli strain that was not able to escape from engineered biocontainment before, and we find that it does not escape even after 100 days of evolution, nor does it escape when added to mammalian cell culture.

synthetic biology↗

bioRxiv2019

Computational design of a modular protein sense/response system

Sensing and responding to signals is a fundamental ability of living systems, but despite remarkable progress in computational design of new protein structures, there is no general approach for engineering arbitrary new protein sensors. Here we describe a generalizable computational strategy for designing sensor/actuator proteins by building binding sites de novo into heterodimeric protein-protein interfaces and coupling ligand sensing to modular actuation via split reporters. Using this approach, we designed protein sensors that respond to farnesyl pyrophosphate, a metabolic intermediate in the production of valuable compounds. The sensors are functional in vitro and in cells, and the crystal structure of the engineered binding site matches the design model with atomic accuracy. Our computational design strategy opens broad avenues to link biological outputs to new signals.\n\nOne Sentence SummaryAn engineering strategy to design modular synthetic signaling systems that respond to new small molecule inputs.

biophysics↗