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In the field of synthetic biology, genetic engineering techniques are used to create modular components and novel biological interactions within microorganisms. One example of this is the development of toggle switches, which are bistable genetic circuits that can store 1 bit of information in living organisms like E. coli bacteria. However, existing toggle switches based on promoter-repressor pairs, such as LacI and TetR, have limitations in terms of orthogonality and programmability. Recent advancements in synthetic biology have demonstrated the potential of using catalytically inactive versions of Cas proteins, known as dCas, to create logic switches. These dCas proteins can selectively bind to specific DNA sequences and offer an alternative approach for constructing functional toggle switches, addressing the limitations of traditional designs. In this study, we aim to utilize the CRISPRi-dCas12a system as the foundation for our genetic toggle switch. To begin, we develop a thermodynamic model to explore the factors influencing the repression efficiency of CRISPRi-dCas12a. We assume that the fully assembled dCas12a-crRNA complex functions similarly to the simple repression motif regulatory architecture when targeting the promoter region, creating a basic genetic NOT gate. Our model reveals that the competition between RNAP and the dCas12a complex for binding to the same promoter, as well as the availability and affinity of target sites and potential competitor sites, all contribute to the strength of promoter occupancy competition. To experimentally investigate the concealed thermodynamic factors affecting Cas12a DNA binding, we employ a high-throughput assay called XSeq. This assay allows us to examine various parameters, including the impact of protospacer adjacent motif sequences, the type and location of guide-target mismatches, and their influence on Cas12a DNA binding. Next, we construct our CRISPRi toggle switch by interconnecting two of the aforementioned NOT gates in a mutually inhibitory network. To understand the bistability of CRISPRi toggle switches at the steady state, we develop a thermodynamic model that considers parameters such as target sequence affinity, crRNA properties, net production activity of the dCas12a complex, and the copy number of the toggle switch. We then leverage the XSeq assay to efficiently characterize these parameters for numerous toggle switch constructs. Furthermore, to visualize the kinetics of our CRISPR-based toggle switch designs, we introduce a second controller plasmid to set and reset the switch state. By combining single-cell imaging techniques with a microfluidic chip platform, we can observe the real-time actuation dynamics of the toggle switch. Our results uncover different classes of switching activities and memory retention abilities, demonstrating how manipulating the growth conditions of the bacteria through different media types can influence the behavior of the toggle switch.

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197 pages


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CRISPR-dFnCas12a; CRISPRi; genetic circuits; Toggle switch; Tunable plasmid copy number


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Lambert, Guillaume

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Brito, Ilana
De Vlaminck, Iwijn

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Ph. D., Biophysics

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Doctor of Philosophy

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Government Document




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dissertation or thesis

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