Phosphorylation based insulation devices design and implementation

Abstract

This thesis presents the analysis of a phosphorylation based insulation device implemented in Saccharomyces cerevisae and the minimization of the retroactivity to the input and retroactivity to the output of a single cycle phosphorylation device by means of optimal substrate and phosphatase concentration selection. Characterizing and improving the performance of insulation devices brings us a step closer to their successful implementation in biological circuits, and thus to modularity. To this end, an insulation device was designed and implemented in Saccharomyces cerevisae employing the principle of timescale separation. It was shown experimentally (data pending publication), that the dynamics of the insulation device output remained unchanged in the presence of promoter sites (load) providing retroactivity. In this thesis, the underlying mechanism by which the insulation device retains its dynamic performance in the presence of load is explained through singular perturbation and parameter sensitivity analysis. It was determined that the fast phosphotransfer reactions of the insulation device indeed allowed for retroactivity attenuation provided the substrate and phosphatase concentration are in sufficient amounts. Furthermore, the retroactivity to the input and retroactivity to the output of phosphorylation based insulation devices were parameterized with the substrate and phosphatase concentrations using a single cycle model. While previous works have focused on showing output retroactivity attenuation through high substrate and phosphatase concentration, it is shown that this has detrimental effects on the insulation device performance even in isolation. Employing singular perturbation and contraction theory tools, this work provides a framework to determine an optimal substrate and phosphatase concentration to reach a tradeoff between the retroactivity to the input and the retroactivity to the output.