(277e) A One-Step Procedure for the Optimal Connection of Synthetic Genetic Circuits

Engineering complex genetic circuits remains a time-consuming and laborious affair. These systems combine multiple genetic elements, such as promoters, ribosome binding sites, and protein coding sequences, to create interesting and potentially useful dynamical or logical behaviors. While individual genetic circuits may function as expected, successfully connecting them together to create higher-order behaviors often requires significant trial-and-error mutagenesis. Connections are made by having the output of one genetic circuit, typically a transcription factor, regulate the input of another genetic circuit. A key challenge is determining the production rates of the inputs and outputs such that their overall regulatory behavior exhibits the desired dynamics or logic.

Here, we present a formal design procedure for functionally connecting synthetic genetic circuits together, requiring only a single genetic modification to the original system. First, the method identifies the optimal production rates of the inputs and outputs of two genetic circuits, such that the systems behavior exhibits a desired logical behavior. Second, the procedure generates a specific DNA sequence that will enact these changes. To do this, the design procedure has three ingredients: (i) the computational predictions of the ribosome binding site (RBS) calculator¹, a newly developed predictive design method for ribosome binding sites; (ii) the transfer function data for each synthetic genetic circuit, quantifying the steady-state relationship between their inputs and outputs; and (iii) the original DNA sequence of the two synthetic genetic circuits. The user defines a fitness function describing the desired logical behavior and the procedure uses these three ingredients to identify the optimal sequence of the regulatory protein's ribosome binding site that maximizes the value of the fitness function. Inserting this ribosome binding site sequence upstream of the protein will result in the successful connection of two synthetic genetic circuits with an optimal higher-order behavior.

To demonstrate its utility, we employ the design procedure to functionally connect together two synthetic genetic circuits. As an example, we connect a pair of signal-responsive inputs to a previously constructed AND logic circuit². Without genetic modification, the production rates of the inputs are either too high or too low, causing the output of the logic circuit to poorly exhibit AND logic. Given a fitness function describing AND logic, the design procedure predicts the sequence of a ribosome binding site such that the correct AND logic is outputted. We experimentally verify that the generated sequence results in near-optimal AND logic by characterizing the combined system. We also rationally perturb the input rates to measure the systems's deviation from optimality.

¹Salis H, Mirsky EA, Voigt CA, "Automated Design of Synthetic Ribosome Binding Sites to Precisely Control Protein Expression", (in review)

²Anderson JC, Voigt CA, Arkin AP, "Environmental signal integration by a modular AND gate", Nat. Mol. Syst. Biol., 2007