J66688 mb

All succinic acid production strains were picked into 6 mL of synthetic minimal (SD) medium and grown at 30 °C, 250 rpm overnight. The next day, optical density was measured in a spectrophotometer (WPA Biowave II) and cultures were diluted to OD₆₀₀ = 0.05 in 1 mL SD media, with and without 1 µM aTc (Alfa Aesar, J66688-MB). Cultures were grown in 48-deep-well-plates (Agilent, 201238-100) at 30 °C in an Infors HT Multitron, shaking at 700 rpm. After 2 days, plates were spun down at 4000 × g, 4 °C for 10 min. Then, 300 µL of the supernatant was sampled for each well. The same day, supernatant samples were measured directly by LC-MS alongside a succinic acid standard, as follows: succinic acid was detected and measured by UPLC-MS, using an Agilent 1290 Affinity chromatograph linked to an Agilent 6550 Q-ToF mass spectrometer. Separation was achieved using an Agilent Zorbax Eclipse Plus C18 column (2.1 × 50 mm, 1.8 μm) and an acetonitrile gradient of 0% for 2 min then an increase to 98% over 0.5 min at a flow rate of 0.3 mL/min. Mass spectral data was acquired in negative ion mode from m/z 90 to 1000 at the rate of 3 spectra per second throughout the separation. In total, 0.2 µL was injected from both sample wells and standard solutions. Succinic acid concentrations were calculated from a succinic acid standard curve in Microsoft Excel.

All reporter strains were picked into 500 μL of synthetic complete (SC) medium and grown in 2.2 mL 96 deep-well plates at 30 °C in an Infors HT Multitron, shaking at 700 rpm overnight. The next day, saturated strains were diluted 1:100 into fresh media, with and without 1 µM aTc (Alfa Aesar, J66688-MB). For single-point measurements, cultures were incubated for 16 h and cell fluorescence was measured by an Attune NxT Flow Cytometer (Thermo Scientific). Batch culture and daily cell passaging assay experiments as described in the text. Attune NxT Flow Cytometer settings: FSC 300 V, SSC 350 V, BL1 500 V, VL2 450 V, YL2 450 V. Fluorescence data was collected from at least 10,000 cells for each experiment and analysed using FlowJo software. Note: 1 µM (463 ng/µL) aTc was used, rather than the standard 100 ng/µL, to ensure ligand saturation and full release of the mutTetR-Mxi1 protein from the array. 1000 x stock solution of aTc (1 mM) was in 100% DMSO. Final concentration of DMSO was present in all conditions.

All quantitative PCR (qPCR) strains were picked into 5 mL of synthetic complete (SC) medium and grown at 30 °C, 250 rpm overnight. The next day, optical density was measured in a spectrophotometer (WPA Biowave II) and cultures were diluted to OD₆₀₀ = 0.05 in 5 mL SC media, with and without 1 µM aTc (Alfa Aesar, J66688-MB). For RNA purification, RNA was isolated from yeast culture grown to an OD₆₀₀ of 1 ± 0.1 using a RiboPure Yeast kit (Invitrogen). RNA was quantified by nanodrop spectrophotometer (Thermo Fisher), and cDNA was generated from each RNA prep using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Each qPCR reaction contained 10 ng of cDNA. qPCR results were normalized to the housekeeping gene UBC6. All qPCR primers were designed manually using Benchling. All quantitative PCR (qPCR) reactions were performed in an StepOnePlus™ Real-Time PCR System (Applied Biosystems) using SYBR Green JumpStart Taq ReadyMix (Sigma-Aldrich).