Integrase enables synthetic intercellular logic via bacterial conjugation.

Integrases have been widely used in synthetic biology for genome engineering and genetic circuit design. They mediate DNA recombination to alter the genotypes of single cell lines in vivo, with these changes being permanently recorded and inherited via vertical gene transfer. However, integrase-based intercellular DNA messaging and its regulation via horizontal gene transfer remain underexplored. Here, we introduce a versatile strategy to design, build, and test integrase-based intercellular DNA messaging through bacterial conjugation. First, we screened conjugative plasmids and recipient cells for efficient conjugation. Then, we established a layered framework to describe the interactions among hierarchical E. coli strains and implemented dual-layer Boolean logic gates to demonstrate intercellular DNA messaging and management. Finally, we expanded the design to include four-layer single-processing pathways and dual-layer multi-processing systems. This strategy advances intercellular DNA messaging, hierarchical signal processing, and the application of integrase in systems and synthetic biology.