Engineering synthetic notch receptors and activation-inhibition genetic circuits to regulate gene expression levels
Embargo Date
2025-09-11
OA Version
Citation
Abstract
Self-organized pattern formation during organismal development, such as skin patterning and lung branching, may be explained by a reaction-diffusion model with only two diffusible morphogens. The core signaling circuit in the proposed model comprises activating and repressing pathways, through which an activator and an inhibitor regulate the expression of one another. It has been challenging to extract the minimal requirements and study the dynamics of the activation-inhibition networks in natural biology systems because they are embedded within other gene regulation and signaling pathways. Recapitulating the genetic circuits and regulatory relationships of the model with synthetic components would allow us to control and characterize their behavior and ultimately allow us to implement more complex multicellular communication. This thesis aimed to engineer and investigate synthetic genetic circuits to regulate gene expression by activation and repression in mammalian cells. To induce changes in gene expression in response to arbitrary extracellular molecules, cells were engineered with modular synthetic Notch (synNotch) transmembrane receptors that created a novel signaling pathway. The activation circuits allowed cells equipped with synNotch receptors to recognize fluorescent proteins that were either membrane-tethered or caught by anchors on a neighboring sender cell and induce downstream gene expression. Next, I investigated repression circuits mediated by three molecular engineering tools: chromatin regulators (CR), antisense promoters, and site-specific endoRNAse. I engineered the intracellular domain of the synNotch receptors to construct each repressor circuit and measured the decrease in reporter expression to compare their performance. Lastly, components of the activator and repressor circuits were combined to engineer a bipotential cell population that responds to morphogens to regulate gene expression in both directions in response to activation or repression. In the future, optimizing the circuits and integrating feedback circuits regulated by morphogens with controllable diffusion rate may help us to mimic higher-order complex multicellular behavior, like the Turing pattern or other reaction-diffusion self-organizing models.
Description
License
Attribution-NonCommercial-ShareAlike 4.0 International