Synthetic biomolecular control systems to reprogram cellular functions and dynamic responses

Chen, WilfredLiving systems of all scales must constantly adapt to changing conditions by surveying their surroundings and executing appropriate responses. To adequately respond to the diverse environmental stimuli presented by nature, living organisms have evolved a vast repertoire of molecular circuits to direct their physiological behaviors. In contrast, synthetic biological systems are not yet equipped with robust, autonomous, and generalizable biomolecular regulatory elements to implement dynamic control circuits necessary for advanced biotechnology and human health applications. There is, thus, a need to develop new biomolecular tools to mimic and integrate various aspects of natural control systems into functional regulatory schemes. ☐ The first step in any dynamic control scheme involves the recognition of a given stimuli by a sensor. While numerous sensing machineries have been found in nature that provide a rich toolkit of sensory components for many forms of stimuli, these naturally occurring components are not adapted for use in an artificial context. To bypass the often tedious process of engineering membrane receptors and intracellular signaling pathways, we envisioned a synthetic extracellular sensing circuit that can exploit pre-existing membrane receptors by input-induced reconstitution of native signaling peptide. The biochemical basis of such a sensing circuit was established by adapting intein-mediated reactions to reconstitute the well-known yeast mating pheromone peptide, α-factor, and exploiting the associated yeast mating pathway to direct cellular responses. ☐ Biological control systems contain logic elements that receive and process signals from sensory elements to decide what responses need to be executed. A particular hurdle for integrating synthetic components into control circuits is the prevalence of unspecific interactions and crosstalk that inevitably arise from the crowded cellular milieu. In addition, the inability to utilize endogenous cellular information as inputs for activating or repressing responses prohibit the use of synthetic control elements from being used in autonomous regulatory schemes. These limitations drove the design and construction of a new class of riboregulators, termed toehold-gated guide RNA. The synthetic riboregulators can be programmed to respond to a very large variety of RNA sequences, including full-length mRNA, and control CRISPR/Cas9 activities for multiplexed gene regulation in E. coli with minimal cross-talk. The versatility of this platform was demonstrated by the use of endogenous RNA transcripts as triggers to activate Cas9 functions, allowing thgRNA to be used as an autonomous control elements in dynamic control schemes. ☐ Molecular control circuits rely on precise protein-protein interactions to be made consistently to execute the appropriate responses. The actuations of biomolecular responses are tightly regulated by post-translational modifications such as phosphorylation, which are not readily applicable to synthetic output components like heterologous enzymes or transcription factors. One way to circumvent the use of post-translational modifications to control protein-protein interactions is to apply coiled-coil motifs as recombinant peptide tags on synthetic components to direct their interactions. Dynamic reconfiguration of protein complex can be achieved by taking advantage of coiled-coil motifs and their associated strand displacement reaction. Control over a heterologous enzyme complex and its productivity was demonstrated.University of Delaware, Department of Chemical and Biomolecular EngineeringPh.D