Fusco, Virginia (2024) Engineering Biomolecular Circuits for Advanced Cell-Based Bioproduction and Biosensing. [Tesi di dottorato]

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Abstract

Advances in biotechnological tools have enabled precise control over cellular behavior by allowing cells to express exogenous genes ”on demand” via specific genetic constructs which respond to treatment with inducer molecules. However, cellular engineering often lacks advanced regulatory mechanisms, resulting in designs that may not perfectly align with intended functional objectives. As a result, developing engineered cells can be a time-consuming process, requiring multiple iterations to adjust biological components until the desired cell behavior is achieved. Integrating control engineering principles into this process offers a promising solution for achieving precise gene regulation, reducing the need to re-engineer individual components by using predictable control actions to correct unwanted behaviors. In this work, I engineered biomolecular circuits to achieve highperformance inducible gene expression systems by means of two regulatory architectures. The first is a modified feed-forward loop controller designed to minimize basal expression in the absence of an inducer molecule and to maximise gene expression in its presence. The second employs an antithetic integral controller, allowing for precise and robust regulation of the input-output dynamics in gene expression, while also improving system linearity and responsiveness. Thus, I designed, modelled and experimentally implemented biomolecular circuits that combine these architectures in different ways to create high-performance inducible gene expression systems. I then demonstrated their adaptability in various practical applications, such as improving production of Adeno Associated Viral (AAV) vectors, or as a biosensor which monitors in vitro and in vivo key biological signals. These examples highlight the potential of integrating synthetic biology with control engineering, offering a direct pathway from theoretical concepts to practical applications in gene therapy, paving the way for innovative solutions in the field.

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