Exploiting natural chemical photosensitivity of anhydrotetracycline and tetracycline for dynamic and setpoint chemo-optogenetic control

The transcriptional inducer anhydrotetracycline (aTc) and the bacteriostatic antibiotic tetracycline (Tc) are commonly used in all fields of biology for control of transcription or translation. A drawback of these and other small molecule inducers is the difficulty of their removal from cell cultures, limiting their application for dynamic control. Here, we describe a simple method to overcome this limitation, and show that the natural photosensitivity of aTc/Tc can be exploited to turn them into highly predictable optogenetic transcriptional- and growth-regulators. This new optogenetic class uniquely features both dynamic and setpoint control which act via population-memory adjustable through opto-chemical modulation. We demonstrate this method by applying it for dynamic gene expression control and for enhancing the performance of an existing optogenetic system. We then expand the utility of the aTc system by constructing a new chemical bandpass filter that increases its aTc response range. The simplicity of our method enables scientists and biotechnologists to use their existing systems employing aTc/Tc for dynamic optogenetic experiments without genetic modification.


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Methodology
Sample preparation Instrument Software Cell population abundance Gating strategy Each experiment in the main text was performed in triplicates and is on par with standard bacterial experiments reported in the field.
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Fluorescence measurements: Cells grown in minimal media were sampled and mixed with the same volume of 500 !g/ ml rifampicin and 50 µg/ ml tetracycline in phosphate-buffered saline. mCherry fluorescence was matured for 90 min at 37°C and read undiluted on the flow cytometer. Cell count measurements: 100 !l samples of cells grown in minimal media were mixed with 79 !l of 500 !g/ ml rifampicin and 50 µg/ml tetracycline in phosphate-buffered saline and 21 !l of 2 !m AccuCount Blank Particles (Spherotech) before being measured on a flow cytometer. mCherry fluorescence was measured with a 561 nm laser and 610/20 nm band pass filter and following gain settings: forward scatter 100, side scatter 100, mCherry gain 1,500 when mCherry was expressed from aTc-regulated promoters and 300 gain when mCherry was expressed with Opto-T7RNAP due to the difference in expression levels. Thresholds of 2,500 FSC-H and 1,000 SSC-H were used for all samples. The flow cytometer was calibrated before each experiment with QC beads (CytoFLEX Daily QC Fluorospheres, Beckman Coulter) to ensure comparable fluorescence values across experiments from different days. At least