Alexa N. Gormick, Adam M. Zahm, Samuel R. Himes, Kathleen E. Rondem, Justin G. English
Chemogenetic regulators of transgene activity, such as the tetracycline-inducible system derived from the tetracycline resistance operon of the bacterial transposon Tn10, are critical and widely used systems in cellular engineering. The tetracycline-inducible system is prized for its selectivity, high affinity, inducibility, reversibility, and differential control of gene transcription. However, its optimization for binary on/off expression limits its application in systems biology and the modeling and construction of complex regulatory systems with intricate input/output paradigms. To overcome this limitation, we developed a high-throughput reporter system to investigate a saturated mutagenesis library of tetracycline resistance operator variants. Using this system, we mapped the functional interactions of Tet repressor DNA binding protein at single-nucleotide resolution in mammalian cells. Our comprehensive screen revealed a spectrum of variant effects, ranging from a nearly complete loss of repression to levels indistinguishable from the natural operator, validated through orthogonal assays. This comprehensive characterization of the sequence-specificity of a tetracycline resistance operator facilitates the construction of variably suppressive, inducible systems for dynamic and modular control over gene expression in mammalian cell culture.
Alexa N. Gormick, Adam M. Zahm, Samuel R. Himes, Kathleen E. Rondem, Justin G. English. High-Throughput Characterization of Tetracycline Repressor Function on Tetracycline Operator 2 Variants. 2025-06-09. https://doi.org/10.1021/acssynbio.4c00809
An episomal pcDNA3.1-tetO-BC-mGL library containing 96 designed tetO2 operators and approximately 100 random 24-nt barcodes per operator was assayed in HEK293 cells. Variant-level TetR repression was measured by comparing RNA/DNA reporter activity with TetR present under untreated versus doxycycline-treated conditions, with a mGreenLantern co-transfection arm as a repressor-independent control.
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