High-Throughput Characterization of Tetracycline Repressor Function on Tetracycline Operator 2 Variants

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.