Synthetic genetic circuits have revolutionized numerous fields, ranging from academic research and point-of-care diagnostics to disease therapeutics and industrial biomanufacturing. These circuits provide a powerful tool for precise spatiotemporal control over biological and biochemical interactions, thereby enhancing our understanding of these complex systems and expanding their applicability. The last few decades have witnessed a surge in research efforts, both in cell-free and cellular systems. These endeavors include those to improve the sensitivity and specificity of diagnostics and optimize the safety, efficacy, and tunability of existing treatments. This dissertation delves into the exploration of Boolean logic gates in the cell-free realm: the development of a 'Cell-Free Recombinase Integrated Boolean Operating System' (CRIBOS) for expanding the capabilities of cell-free sensing applications.
Applications of Boolean logic gates have flourished within cellular systems and animal models. However, a persisting gap in the field is in their exploration within the cell-free system. This deficiency has resulted in a constrained toolkit for studying and applying Boolean logic gates in cell-free settings. Recognizing this limitation in the field and aiming to extend the frontiers of genetic circuits beyond traditional boundaries, I introduce CRIBOS, leveraging the advantages of recombinase, known for its high orthogonality, efficiency, and sensitivity. I designed more than 20 multi-input-multi-output recombinase Boolean logic gates in a cell-free context, from which a set of critical rules crucial for building genetic circuits in the cell-free environment was also established. In addition, integrating allosteric transcription factor (aTF)-based sensors with CRIBOS enabled multiplex environmental sensing within the cell-free environment. Moreover, the CRIBOS system showcased its versatility by facilitating the creation of a biological memory storage device, demonstrating robust functionality with high stability over four months. Implementing CRIBOS not only expands the application of multiplex Boolean logic gates from cellular systems to the cell-free environment but also expands their overall versatility, opening new avenues for the design and application of sophisticated genetic circuits.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/48850 |
| Date | 23 May 2024 |
| Creators | Chen, Jingyao |
| Contributors | Wong, Wilson W. |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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