G·A·B·B·A

Synthetic biology is a flourishing discipline that holds great promise; by attempting to build biological systems we can test the limits of our understanding. In the last decade, the field has advanced a great deal with several synthetic gene circuits being developed, including genetic switches, oscillators, cell-cell communication modules and multicellular networks, although the vast majority were built in bacteria and yeast. This thesis establishes the design, construction and characterization of an artificial synthetic cell-cell communication system in mammalian cells. This was used to engineer spatial patterns: gradients of reporter activation or repression over fields of cells. The model system used was the Madin-Darby canine kidney (MDCK) cell line, which forms a polarized monolayer enclosing a fluid-filled lumen (cysts) when embedded in a collagen matrix. These cysts were used as a scaffold for engineering synthetic pattern-forming gene networks, in a three dimensional context. For this I initially developed a method to transfect MDCK cysts locally, achieving distinct regions of transfection. I then built and characterized a fluorescent reporter gene construct that is induced by hepatocyte growth factor and repressed by a truncated variant, NK4. Finally, I engineered a synthetic spatial patterning platform for widefield microscopy, based on localised expression-secretion of HGF or NK4. The former diffuses from sender cells and modulates tubule formation and fluorescent reporter gene expression in the receiver cells, whilst the latter represses this effect.