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Abstract The field of synthetic biology and biosystems engineering increasingly acknowledges the need for a holistic design approach that incorporates circuit-host interactions into the design process. Engineered circuits are not isolated entities but inherently entwined with the dynamic host environment. One such circuit-host interaction, ‘growth feedback’, results when modifications in host growth patterns influence the operation of gene circuits. The growth-mediated effects can range from growth-dependent elevation in protein/mRNA dilution rate to changes in resource reallocation within the cell, which can lead to complete functional collapse in complex circuits. To achieve robust circuit performance, synthetic biologists employ a variety of control mechanisms to stabilize and insulate circuit behavior against growth changes. Here we propose a simple strategy by incorporating one repressive edge in a growth-sensitive bistable circuit. Through both simulation and in vitro experimentation, we demonstrate how this additional repressive node stabilizes protein levels and increases the robustness of a bistable circuit in response to growth feedback. We propose the incorporation of repressive links in gene circuits as a control strategy for desensitizing gene circuits against growth fluctuations. 

The effects of host resource limitations on the function of synthetic gene circuits have gained significant attention over the past years. Hosts, having evolved resource capacities optimal for their own genome, have been repeatedly demonstrated to suffer from the added burden of synthetic genetic programs, which may in return pose deleterious effects on the circuit’s function. Three resource controller archetypes have been proposed previously to mitigate resource distribution problems in dynamic circuits: the local controller, the global controller, and a “negatively competitive” regulatory (NCR) controller that utilizes synthetic competition to combat resource competition. The dynamics of negative feedback forms of these controllers have been previously investigated, and here we extend the analysis of these resource allocation strategies to the incoherent feedforward loop (iFFL) topology. We demonstrate that the three iFFL controllers can attenuate Winner-Take-All resource competition between two bistable switches. We uncover that the parameters associated with the synthetic competition in the NCR iFFL controller are paramount to its increased efficacy over the local controller type, while the global controllers demonstrate to be relatively ineffectual. Interestingly, unlike the negative feedback counterpart topologies, iFFL controllers exhibit a unique coupling of switch activation thresholds which we term the “coactivation threshold shift” effect. Finally, we demonstrate that a nearly fully orthogonal set of bistable switches could be achieved by pairing an NCR controller with an appropriate level of controller resource consumption. 

The mutual interactions between the synthetic gene circuits and the host growth could cause unexpected outcomes in the dynamical behaviors of the circuits. However, how the steady states and the stabilities of the gene circuits are affected by host cell growth is not fully understood. Here, we developed a mathematical model for nonlinear growth feedback based on published experimental data. The model analysis predicts that growth feedback could significantly change the qualitative states of the system. Bistability could emerge in a circuit without positive feedback, and high-order multistability (three or more steady states) arises in the self-activation and toggle switch circuits. Our results provide insight into the potential effects of ultrasensitive growth feedback on the emergence of qualitative states in synthetic circuits and the corresponding underlying mechanism.