Exploring the design principles of orthgonal transcription control systems

The last two decades has witnessed an unprecedented growth in our ability to engineer biological systems for a wide range of applications ranging from the development of smart therapeutics, production of valued products and chemicals and engineering crops with programmable traits and much more. At the core of these capabilities has been the design and characterization of synthetic genetic programs that has enabled the predictable programming of cellular behavior and phenotypes. A fundamental challenge in the construction of such circuits and programs is being able to design and model them against a variety of organismal backgrounds, which can be often difficult to predict and can lead to circuit failure when systems are ported across organisms. Such failure modes can potentially be mitigated by embedding orthogonal modes of transcriptional control and regulation in genetic programs to drive the expression of the circuit components in both prokaryotes as well as eukaryotes. Specifically, in prokaryotes, we demonstrate how an autoregulated network controlling the expression of an orthogonal RNA polymerase – T7 RNA polymerase, can be utilized to precisely express target genes in a highly predictable manner dictated by mutant T7 RNAP promoters. Furthermore, with the use of a modular architecture we show how such expression systems can be readily ported across diverse prokaryotes. In each species, the relative strength of expression obtained from the T7 RNAP homeostasis circuit is nearly identical, suggesting T7 RNAP driven expression systems can be utilized as predictable cross-species gene expression platform. In another example, orthogonal transcriptional regulation was engineered in a complex eukaryote (plants) using a programmable transcription factor - dCas9:VP64 and a set of designed synthetic promoters whose activity can precisely regulated with the expression of specific guide RNAs (gRNAs). This strategy was used to construct three mutually orthogonal promoters, allowing multiplexed control of gene expression in plants. Overall, the design strategies and architectures described in this work can be used to explore the design of more complex circuits where the activity of T7 RNAP can be coupled to regulate the activity of dCas9 based transcription to generate circuits operating across kingdoms of life.