Fig. 4: Building programmable protein switches using GPlad and antiGPlad.
From: De novo designed protein guiding targeted protein degradation
a Schematic representation of the reversible switch. b Degradation and accumulation of mKate2 controlled by the reversible switch. Initially, mKate2 expression was induced for 2 h by adding 0.1 mM IPTG (t = 0 h time point). Subsequently, 20 ng/mL aTc was added to induce GPlad, leading to the degradation of mKate2. Finally, 1% arabinose was added to induce antiMcsB, thereby halting mKate2 degradation and allowing for its gradual accumulation. c Flow cytometry was used to continuously monitor the degradation and accumulation of mKate2 under the control of a reversible protein switch. d Designed complexes of GPLacI-LacI, with binding sites (yellow) and targets (gray). e Schematic representation of an amplifier. The LacI and mKate2 were expressed from the genome, and the GP-McsB was expressed from plasmid pGPlad. f Evaluation of the minimum IPTG induction concentration threshold with the assistance of an amplifier in E. coli. Corresponding cells harboring the amplifier were treated with different concentrations of aTc to detect mKate2 fluorescence by a microplate reader. g Schematic representation of an oscillator. The LacI and mKate2 were expressed from the genome, while the GP-McsB and antiMcsB were expressed from plasmid pAntiGPlad. h Optimization of the antiMcsB promoter to achieve oscillatory changes in intracellular mKate2 abundance. Data represent mean ± s.d. from three independent experiments. i Oscillation curve of mKate2. The addition of 20 ng/mL aTc at 6 h extended the oscillation period to 14 h. All experiments are presented as mean ± s.d. from three biologically independent replicates. Source data are provided as a Source Data file. (a, e, g) are created in BioRender https://BioRender.com/912hekk.
