Cumate-Induced Gene Expression Monitoring

Cultures of strain UJP505 or UJP209 carrying pQFT-mNG were grown overnight in LB-Lennox supplemented with oxytetracycline at 37°C, diluted 1:1,000 in the same medium, and grown for another 3 h at 37°. Amounts of 2 μL of cultures were spotted on LB-Lennox 1% agarose pads containing oxytetracycline with or without 600 μM cumate, and images were acquired every 5 min using a DeltaVision system with softWoRx 6.0 (GE Healthcare) on an Olympus IX71 microscope equipped with a pco.edge scientific complementary metal oxide semiconductor (sCMOS) camera and an UPlan FL N 100×/1.30 oil objective (Olympus) at 37°C. The mNeonGreen signal was recorded using standard green fluorescent protein (GFP) excitation and emission filters with 100-ms exposure time at 50% power. The ImageJ plugin MicrobeJ (58 (link)) was used for quantification of single-cell fluorescence signals on time-lapse data from three biological replicates with two fields of view each.