AbstractModern genetic tools allow the dissection and emulation of fundamental mechanisms shaping morphogenesis in multicellular organisms. Several synthetic genetic circuits for control of multicellular patterning have been reported to date. However, hierarchical induction of gene expression domains has received little attention from synthetic biologists, despite its importance in biological self-organization. We report the first synthetic genetic system implementing population-based AND logic for programmed autonomous induction of bacterial gene expression domains. We develop a ratiometric assay for bacteriophage T7 RNA polymerase activity and use it to systematically characterize different intact and split enzyme variants. We then utilize the best-performing variant to build a three-color patterning system responsive to two different homoserine lactones. We validate the AND gate-like behavior of this system both in cell suspension and in surface culture. Finally, we use the synthetic circuit in a membrane-based spatial assay to demonstrate programmed hierarchical patterning of gene expression across bacterial populations.Abbreviations3OC6HSLN-(3-oxohexanoyl)-L-homoserine lactone3OC12HSLN-(3-oxododecanoyl)-L-homoserine lactoneCFPcyan fluorescent proteinIPTGisopropyl-β-D-thiogalactopyranosideParts RegistryMIT Registry of Standard Biological PartsPIpositional informationRDreaction-diffusionRFPred fluorescent proteinRFUrelative fluorescence unitss.d.standard deviationT7RNAPbacteriophage T7 RNA polymeraseYFPyellow fluorescent protein
A self-initiating eukaryotic transient gene expression system based on cotransfection of bacteriophage T7 RNA polymerase and DNA vectors containing a T7 autogene
