Synthetic Circuits for Feedback and Detection in Bacteria

Synthetic biology, by co-opting molecular machinery from existing organisms, can be used as a tool for building new genetic systems from scratch, for understanding natural networks through perturbation, or for hybrid circuits that piggy-back on existing cellular infrastructure. Although the toolbox for genetic circuits has greatly expanded in recent years, it is still difficult to separate the circuit function from its specific molecular implementation. In this thesis, we discuss the function-driven design of two synthetic circuit modules, and use mathematical models to understand the fundamental limits of circuit topology versus operating regimes as determined by the specific molecular implementation. First, we describe a protein concentration tracker circuit that sets the concentration of an output protein relative to the concentration of a reference protein. The functionality of this circuit relies on a single negative feedback loop that is implemented via small programmable protein scaffold domains. We build a mass-action model to understand the relevant timescales of the tracking behavior and how the input/output ratios and circuit gain might be tuned with circuit components. Second, we design an event detector circuit with permanent genetic memory that can record order and timing between two chemical events. This circuit was implemented using bacteriophage integrases that recombine specific segments of DNA in response to chemical inputs. We simulate expected population-level outcomes using a stochastic Markov-chain model, and investigate how inferences on past events can be made from differences between single-cell and population-level responses. Additionally, we present some preliminary investigations on spatial patterning using the event detector circuit as well as the design of stationary phase promoters for growth-phase dependent activation. These results advance our understanding of synthetic gene circuits, and contribute towards the use of circuit modules as building blocks for larger and more complex synthetic networks.

Synthetic regulation of eukaryotic gene expression by noncoding RNA

Synthetic biological systems promise to combine the spectacular diversity of biological functionality with engineering principles to design new life to address many pressing needs. As these engineered systems advance in sophistication, there is ever-greater need for customizable, situation-specific expression of desired genes. However, existing gene control platforms are generally not modular, or do not display performance requirements required for robust phenotypic responses to input signals. This work expands the capabilities of eukaryotic gene control in two important directions.

For development of greater modularity, we extend the use of synthetic self-cleaving ribozyme switches to detect changes in input protein levels and convey that information into programmed gene expression in eukaryotic cells. We demonstrate both up- and down-regulation of levels of an output transgene by more than 4-fold in response to rising input protein levels, with maximal output gene expression approaching the highest levels observed in yeast. In vitro experiments demonstrate protein-dependent ribozyme activity modulation. We further demonstrate the platform in mammalian cells. Our switch devices do not depend on special input protein activity, and can be tailored to respond to any input protein to which a suitable RNA aptamer can be developed. This platform can potentially be employed to regulate the expression of any transgene or any endogenous gene by 3’ UTR replacement, allowing for more complex cell state-specific reprogramming.

We also address an important concern with ribozyme switches, and riboswitch performance in general, their dynamic range. While riboswitches have generally allowed for versatile and modular regulation, so far their dynamic ranges of output gene modulation have been modest, generally at most 10-fold. We address this shortcoming by developing a modular genetic amplifier for near-digital control of eukaryotic gene expression. We combine ribozyme switch-mediated regulation of a synthetic TF with TF-mediated regulation of an output gene. The amplifier platform allows for as much as 20-fold regulation of output gene expression in response to input signal, with maximal expression approaching the highest levels observed in yeast, yet being tunable to intermediate and lower expression levels. EC50 values are more than 4 times lower than in previously best-performing non-amplifier ribozyme switches. The system design retains the modular-input architecture of the ribozyme switch platform, and the near-digital dynamic ranges of TF-based gene control.

Together, these developments suggest great potential for the wide applicability of these platforms for better-performing eukaryotic gene regulation, and more sophisticated, customizable reprogramming of cellular activity.

Evolution of developmental gene regulatory networks in echinoids

Developmental gene regulatory networks (dGRNs) are assemblages of regulatory genes that direct embryonic development of animal body plans and their morpho-logical structures. dGRNs exhibit recursively-wired circuitry that is encoded in the genome and executed during development. Alteration to the regulatory architecture of dGRNs causes variation in developmental programs both during the development of an individual organism and during the evolution of an individual lineage. The ex-planatory power of these networks is best exemplified by the global dGRN directing early development of the euechinoid sea urchin Strongylocentrotus purpuratus. This network consists of numerous regulatory genes engaging in hundreds of genomic regulatory transactions that collectively direct the delineation of early embryonic domains and the specification of cell lineages. Research on closely-related euechi-noid sea urchins, e.g. Lytechinus variegatus and Paracentrotus lividus, has revealed marked conservation of dGRN architecture in echinoid development, suggesting little appreciable alteration has occurred since their divergence in evolution at least 90 million years ago (mya).

We sought to test whether this observation extends to all sea urchins (echinoids) and undertook a systematic analysis of over 50 regulatory genes in the cidaroid sea urchin Eucidaris tribuloides, surveing their regulatory activity and function in a sea urchin that diverged from euechinoid sea urchins at least 268 mya. Our results revealed extensive alterations have occurred to all levels of echinoid dGRN archi-tecture since the cidaroid-euechinoid divergence. Alterations to mesodermal sub-circuits were particularly striking, including functional di˙erences in specification of non-skeletogenic mesenchyme (NSM), skeletogenic mesenchyme (SM), and en-domesodermal segregation. Specification of endomesodermal embryonic domains revealed that, while their underlying network circuitry had clearly diverged, regu-latory states established in pregastrular embryos of these two groups are strikingly similar. Analyses of E. tribuloides specification leading to the estab-lishment of dorsal-ventral (aboral-oral) larval polarity indicated that regulation of regulatory genes expressed in mesodermal embryonic domains had incurred significantly more alterations than those expressed in endodermal and ectodermal domains. Taken together, this study highlights the ability of dGRN architecture to buffer extensive alterations in the evolution and early development of echinoids and adds further support to the notion that alterations can occur at all levels of dGRN architecture and all stages of embryonic development.