Aor, I. | Search bioRxiv

bioRxiv2025

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.

synthetic biology↗

bioRxiv2025

Let there be multifunctionality: Uncovering the criticality zoo of the AC-DCgenetic circuit

Gene regulatory networks (GRNs) govern processes such as cell fate, patterning, and adaptation. While multistability and oscillations are both common GRN dynamics in cell biology, they are typically studied and engineered in isolation. Here, we challenge this separation using the AC-DC circuit, a minimal three- gene network that merges the classical toggle switch and repressilator. Using a thermodynamic formalism and Bayesian inference, we show that even a single-inducer version of the circuit can display diverse mul-tifunctional dynamics, including the coexistence of oscillations and multistability. In addition we explore robustness, classify emergent behaviours, and analyse critical slowing down and regime transitions. Re-markably, the AC-DC circuit can produce more than 30 topologically distinct bifurcation diagrams, chal-lenging the classical view that network topology rigidly constrains dynamical outcomes. This flexibility enables synthetic capabilities that couple hysteresis with oscillations, critical slowing down, and reversibility. By uncovering the hidden potential of minimal genetic circuits and outlining design principles for their implementation, this work opens new directions for harnessing emergent complexity using the basic building blocks of life.

systems biology↗