Control of gene expression is essential for synthetic biology. Drug-inducible systems allow for gene expression levels to be precisely regulated through the administration of exogenous chemical inducers, and by combining these systems, more complex circuits with multiple inputs and outputs can be designed. However, the number of existing orthogonal drug-inducible systems is limited. Therefore, there remains a need for new, small-molecule-inducible systems that are orthogonal to both existing systems and native cellular function. Here, we focus on the development and applications of two small-molecule inducible systems for use in basic research, synthetic systems, and therapeutics: the ligand-inducible connection (LInC) system and the HaXS8-inducible system. In the LInC system, the NS3/4a protease from hepatitis C virus is used to regulate the linkage between genetically fused DNA-binding elements and transcriptional effector domains, which remain linked only in the presence of an NS3/4a protease inhibitor. The antiviral drugs which are used as inhibitors for NS3-based systems provide ideal small-molecule inputs for synthetic biology applications, as they are designed to be orthogonal to native biological function and have been tested for clinical use in humans. We demonstrate the construction and validation of a LInC transcription factor (TF) system, as well as further extension of the system with an “all-in-one” single vector design. We then continue on to describe the application of a small molecule (HaXS8) heterodimer system based on the SNAP-tag and HaloTag domains to control gene expression and protein behavior. We demonstrate the construction and validation of several HaXS8-inducible proteins, including TFs, Cre recombinase, and caspase-9. Together, our work on these two systems provides additional orthogonal methods of small molecule-inducible gene expression and protein function for use in control of cellular behavior. / 2024-05-23T00:00:00Z
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/44705 |
| Date | 23 May 2022 |
| Creators | Dotson, Hannah Lin |
| Contributors | Ngo, John T. |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
| Rights | Attribution 4.0 International, http://creativecommons.org/licenses/by/4.0/ |
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