We propose to overcome this problem by integrating rational design with directed evolution. We advocate using rational design (i.e., mathematical modeling) to identify mutational targets for non-functional circuits. Directed evolution can then be used to first systematically perturb the mutational target to generate components with a wide range of functionality, at which point circuit behavior can be evaluated for each component. This procedure builds upon several recent works. Feng et al. also propose using models and random mutagenesis to respectively identify and perturb mutational targets, but suggest screening for functional behavior in the nominal circuit [6]. In contrast, we propose to mutate and characterize these targets with circuits of minimal complexity (i.e., not in the nominal circuit). Characterization of the nominal circuit via systematic perturbation of a single parameter is conceptually similar to the strategy of Alper et al. [1], with the distinction that the targets are not restricted to constitutive promoters. The benefits of this approach are three-fold. First, performing directed evolution on a circuit of minimal complexity ensures that the resulting library covers a wide range of parameter values and facilitates trouble-shooting. Second, the generated library consists of interchangeable components that (1) enable ``plug and play'' alteration of the circuit by simply exchanging one component for another and (2) can readily be applied to other generic circuits. Finally, examining the circuit behavior over a library of components with widely-varying parameter values facilitates model validation and refinement.
We apply this strategy to examine the Lux quorum-sensing module. Natively found in the marine bacterium Vibrio fischeri, a facultative symbiont of luminescent fish or squid, this module regulates gene expression as a function of the population density [7]. The key element of this system is a regulatory cassette consisting of genes encoding LuxI and LuxR. LuxI is an acyl-homoserine lactone (AHL) synthase; LuxR is a transcriptional regulator activated by the AHL. The AHL signal molecule is produced inside the cell, but can freely diffuse across the cell membrane into the environment. Therefore, the AHL concentration is low at low cell density. As the cell density increases, the signal accumulates in the environment and inside the cell. AHL can bind and activate LuxR, activating downstream genes only when the concentration exceeds a threshold due to a positive-feedback mechanism. To better understand this regulatory module, we have constructed design configurations in which different combinations of LuxI and LuxR are controlled through positive feedback. As suggested by a mathematical model, we have probed the possible ranges of system behavior for each of these configurations by employing DNA-binding affinity mutants of LuxR. The altered affinities of these mutants are characterized in a circuit of minimal complexity using a fluorescence-based assay [3]. The results demonstrate that simple recombination events combined with point mutations can readily alter the steady-state characteristics of this module. These results provide a better quantitative understanding of how bacteria regulate cell-cell communication and shed light upon how the natural quorum-sensing configuration might have evolved. We expect these findings to provide insights into controlling pathogens that regulate virulence factors via quorum sensing [10] and facilitate re-engineering of quorum-sensing components for tasks such as cancer therapy [2].