An engineered multi-step differentiation program in Escherichia coli for self-organized spatial patterning

In nature, complex multicellular structures originate from individual cells containing all essential information for differentiation, patterning and morphogenesis. Synthetic biology enables a bottom-up approach to study these processes by engineering and combining individual modules to progressively increase the systems complexity. Here, we engineered a multi-step program mimicking cell differentiation in the model prokaryote Escherichia coli. Starting from genetically identical cells and without providing any external positional information, we generated autonomous spatial patterns of colonies on a solid surface. We first employed a toggle switch to break population homogeneity (symmetry breaking), stochastically differentiating cells into two subpopulations: senders and receivers. Next, we activated expression of a third reporter in receiver colonies located in close proximity to sender colonies via quorum-sensing based communication (paracrine signaling). Finally, we mimic maturation of the newly emerged population by expressing a fourth reporter via an orthogonal, self-activating, quorum sensing signal (autocrine signaling). The diversity of spatial patterns generated by this multi-step program was accurately captured by simulations of a corresponding mathematical model. Together, these results demonstrate that multi-step differentiation programs can be engineered in unicellular bacteria to drive fully self-organized spatial pattern formation.