Numerous gene circuits have been created in the past decade, including bistable switches1-7, oscillators8-10, and logic gates6, 11. Using an integrated approach we engineered an innovative AND gate switch – a synthetic gene regulatory circuit in E. coli. This simple, single promoter, hybrid system is built from well characterized prokaryotic transcriptional building blocks: lac/tet operators and the promoter sequence of ë-phage. It can be tuned with chemical moieties and exhibits an AND gate phenotype with respect to the small-molecule inducers aTc and IPTG. By manipulating the placement of tet and lac operator sequences relative to the -35 and -10 promoter sequences, we successfully generated AND gate functionality at varying (realistic) induction thresholds. Surprisingly, placement of lacO downstream of the Pribnow box in a TTL operator configuration produced the best, most stringent transcriptional control – an interesting phenomenon not previously observed, culminating in improved AND gate performance. Our detailed statistical thermodynamic model of the promoter (of the type employed of Shea and Ackers12) initially predicted the feasibility of the design and quantitatively captured subsequent improvements. This model accurately describes the leakiness of expression as a function of lacO position within the promoter region by employing a Gibbs free energy term describing the energetic “cost” of RNA polymerase binding in the presence of LacI.

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