Engineered Post-Translational Logic (PTL)

Abstract

Current synthetic biological circuits make use of protein-DNA and RNA-RNA interactions to control gene expression in bacteria. Systems that rely on the regulation of gene expression are relatively slow and unsuitable for many applications. Here, we describe our work to engineer synthetic biological systems in yeast using post-translational modifications of proteins to define system state and control cell function; such systems should have faster performance time and enable a wider range of applications. We have specifically chosen to focus on building phosphorylation-driven protein circuits. We modeled a specific instance of a post-translational circuit using methods such as Lyapunov exponents, and showed that the circuit should behave as desired within a large parameter space. We developed a set of peptide tags that can be used to drive the phosphorylation of a chosen substrate by a desired mitogen-activated protein kinase (MAPK). Each phosphorylation event alters a substrate output activity, such as translocation, degradation, or other binding event. These tags were developed using the Phospholocator – a construct whose phosphorylation-mediated translocation is controlled by MAPK activity. Specifically, MAPK phosphorylation of the Phospholocator nuclear localization sequence (NLS) controls recognition of the NLS by cellular import machinery. The Phospholocator serves three purposes: to determine the docking sites of MAPKs of interest, to measure the in vivo activity of such MAP Kinases, and to serve as a first set of post-translational logic parts. Currently, we have built a version of the Phospholocator that is targeted by Cdc28; our next step is to build Fus3-, p38-, and Hog1-activated instances.