Synthetic auxotrophy remains stable after continuous evolution and in coculture with mammalian cells
Aditya M. Kunjapur, Michael G. Napolitano, Eriona Hysolli, Karen Noguera, Evan Appleton, Max G. Schubert, Michaela A. Jones, Siddharth Iyer, Daniel J. Mandell, George M. Church
Abstract
from exhibiting detectable escape from batch cultures, its long-term effectiveness is unknown. Here, we report automated continuous evolution of a synthetic auxotroph while supplying a decreasing concentration of essential biphenylalanine (BipA). After 100 days of evolution, triplicate populations exhibit no observable escape and exhibit normal growth rates at 10-fold lower BipA concentration than the ancestral synthetic auxotroph. Allelic reconstruction reveals the contribution of three genes to increased fitness at low BipA concentrations. Based on its evolutionary stability, we introduce the progenitor strain directly to mammalian cell culture and observe containment of bacteria without detrimental effects on HEK293T cells. Overall, our findings reveal that synthetic auxotrophy is effective on time scales and in contexts that enable diverse applications.