AbstractBuilding synthetic multicellular systems using non‐living molecular components is a grand challenge in the field of bottom‐up synthetic biology. Towards this goal, a diverse range of chemistries have been developed to provide mechanisms of intercellular communication and methods to assemble multicellular compartments. However, building bottom‐up synthetic multicellular systems is still challenging because it requires the integration of intercellular reaction networks with compatible cellular compartment properties. In this study, we encapsulated cell‐free expression systems (CFES) expressing two quorum sensing genetic circuits into droplet interface bilayer (DIB) synthetic cells to demonstrate bidirectional communication. We further develop a method of generating custom DIB multicellular structures by acoustic liquid handling to automatically dispense the CFES droplets and show the potential for multiplexing compartmentalized gene circuits for generating heterogeneous populations of cells. Our work provides a step towards building more complex multicellular systems with intercellular communication from the bottom‐up to study and experimentally model biological multiscalar processes.

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