(F-208) Integrating natural and synthetic patterning systems creates multiscale complexity

Introduction:: Portability of gene circuit is a critical question in synthetic biology. Often a circuit designed in one species is either hard to transplant, or to maintain its original dynamic and functionality without tuning in another species. Among many of the synthetic circuits, pattern formation circuits are known for its high sensitivity, due to the difficulty in precise spatiotemporal control of morphogen, nutrient and other molecules, the complex interaction between parts, and the large and sparse patterning parameter space. Here, we show that a core-ring patterning circuit designed in E.coli can maintain its dynamics in a spontaneous, natural patterning system – swarming Pseudomonas aeruginosa. Not only the patterning dynamic held, the hybrid system also generated de novo patterns at multiple length scale and in different channels. We attribute the portability to the choice of orthogonal genetic elements and seeding configuration. We further explored the seeding setup. Leveraging the cooperation between colonies, the hybrid system developed multi-layered patterns. This work serves as an example of creating complex pattern through modular pattern mechanisms.


Materials and Methods:: The original patterning circuit was constructed in E.coli and published. We combined all parts of the circuit onto a new plasmid with PA14 specific replication origin – PRO1600, and gentamicin resistance gene. This plasmid was transformed into PA14 strains by electroporation. The PA14 strains carrying this circuit were grown on swarming medium, composed of agar, phosphate buffer, salt, casamino acid, and incubated at 37C for 16-48hrs. Time lapse microscopy was used to trace pattern formation in both brightfield and fluorescent channels. We used ImageJ to analyze the imaging results. Chemicals, such as IPTG, 3OC6HSL, 3OC12HSL, were used to tune the patterning dynamics. We also developed mathematical models and solve numerically to predict the pattern formation process.


Results, Conclusions, and Discussions:: In this work, we show patterning gene circuit that thought to be burdensome, sensitive to host physiology and environment can be transplanted into a different species and form patterns at multiple lengthscales and in different channels. The hybrid system also responded as expected to regulatory chemicals, such as quorum sensing molecules and IPTG. By mathematical modeling and liquid culture characterization, we compared the circuit dynamic in PA14 to inside the original E.coli host, the circuit was able to maintain the key requirements for pattern formation. Furthermore, we used liquid dispenser system to control the signaling molecule diffusion. This was shown to be a powerful tool for creating more complex pattern.


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