Mitigating the effects of ribosome limitations on synthetic circuits via high-gain sRNA-mediated negative feedback

Abstract

Resource limitations in bacterial cells can present significant hurdles that preclude correct synthetic circuit behavior. In a simple circuit with one constitutively expressed protein and one protein whose expression is inducible, it has been shown that inducing the expression of the second protein causes a significant decrease in the level of the first. In this thesis, we explore the possibility of reducing the effects of resource limitations by adding a high-gain negative feedback loop to one of the circuits. The loop includes an sRNA construct. We explore different implementations of this circuit and model them mechanistically. Furthermore, we begin physically implementing one of the circuit designs by testing intermediate constructs. Finally, we also explore the hypothesis that exogenous circuits on plasmids compete for a pool of resources that is spatially separated from the resources that the genome utilizes. Through our work, we show results that support the spatial separation hypothesis.