Poster U18 - Microbial teamwork: circuits for in situ differentiation from seed cells

Introduction: Many single-cell organisms exhibit exceptional ability to adapt to environmental perturbations through sophisticated emergent behaviors. Cells evolve myriad strategies to distribute functions within communities to enhance survival in dynamic environments. This division of labor reduces the burden on individual cells, allowing the community to perform diverse tasks efficiently. Inspired by these natural systems, there is great interest in developing robust genetic circuits that can orchestrate distributed tasks across microbial populations. Here, we present a framework for engineering microbial cells to differentiate into multi-fate communities with distributed functions using Synthetic Division of Labor (SynDoL) circuits. The versatility of SynDoL circuits is demonstrated in applications spanning living therapeutics and agricultural biotechnology.

Materials and

Methods: SynDoL circuits use the DNA rearrangement reactions of serine integrases to modulate the state of cells. Cells are initially engineered into a seed state with inactive output promoters. These seed cells have the capability to perform multiple functions but remain quiescent. Upon activation, the SynDoL differentiation circuit employs integrase-logic to stochastically differentiate the circuit to activate only one promoter, leading to the expression of a distinct output function. This study implements SynDoL circuits in the genomes of probiotic E. coli Nissle 1917 and Klebsiella variicola (a rhizosphere-associated endophyte) to evaluate their functionality in vivo.

Results, Conclusions, and Discussions: Cells engineered with SynDoL circuits retained their quiescent seed state across multiple generations, and upon activation, robustly differentiated into a multi-fate community. The differentiation topology can be finely tuned to achieve desired state ratios ranging from 5-90%. In addition, we successfully developed communities with up to four distinct member states and layout a roadmap to scale up the number of states using a combination of orthogonal integrase attachment sequences and ECF sigma factors. To characterize the function of these circuits in vivo, we engineered K. variicola with two- and three-state differentiation circuits. The engineered K. variicola cells can colonize maize roots and differentiate into multi-member communities in situ upon receiving a differentiation cue. Similarly, SynDoL-engineered probiotic E. coli Nissle effectively colonized tumor cores in subcutaneous mouse models and differentiated into multi-fate communities in situ. We anticipate SynDoL circuits to enable engineered cells with enhanced capabilities for sophisticated functions, such as the production of multiple resource-intensive biomolecules in challenging environments, pertinent to addressing pressing challenges in both human health and agriculture sustainability.

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