Boyvat, Dudu (2025) Translational Control by RNA-Binding Proteins in Cell-Free Expression Systems. [Tesi di dottorato]

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Abstract

Cell-free systems provides a cost-effective and versatile platform for biosensor applications, offering rapid responses, simplified workflows, and enhanced stability compared to cell-based systems. However, despite these advantages, their regulatory flexibility (especially at the level of transcriptional repression) remains limited, limiting their dynamic range and robustness. To address this gap, this study investigates post-transcriptional control strategies based on RNA-binding proteins (RBPs) to enhance translational repression in vitro. I designed and characterized genetic circuits regulated by the RBPs MS2-CNOT7 and L7Ae to assess their repression efficiencies in different cell-free systems, including rabbit reticulocyte lysate, E. coli lysate, and the reconstituted cell-free system. MS2-cNOT7 exhibited minimal repression in these contexts, while L7Ae showed measurable but variable repression, particularly in RRL. Based on these findings, I evaluated different genetic architectures and expression protocols in both E.coli lysate and the PURE system. My findings identified that as the most consistent platform for potent RBP-mediated suppression, particularly when combined with optimized backbone design and pre-incubation strategies. Finally, I created an additional layer of translational control capable of responding to viral protease activity by integrating engineered L7Ae to make the protease-responsive module. This work provides a foundation for the development of in vitro protease biosensors and contributes to next-generation diagnostic platforms based on programmable, genetically encoded editing in cell-free systems.

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