Poly d lysine coated glass bottom plates

Cells were plated in poly-D-lysine-coated glass-bottom plates (MatTek Corporation) and switched to phenol-red free culture medium supplemented with 10% FBS prior to live imaging. Cells were imaged using a Nikon Eclipse TE2000 microscope equipped with a chamber for controlled temperature (37%) and CO2 (5%) environment. All live-cell imaging was performed with a 10x Plan Apo objective (Nikon) and a Hamamatsu Orca ER camera using CFP, YFP, mCherry filter sets (Chroma). For EKAREV and EKAREN5 reporter imaging, the FRET signal was collected using customized ECFP/EYFP FRET filter sets with ET436/20x, ET535/30m, and T455lp mounting into the Nikon TE2000/Ti cube.

DNA and cultures were prepared as previously described⁵⁸ . Briefly, the tRNA/Broccoli fusion sequence was cloned into pET30b between the XbaI and BlpI sites downstream of an inducible T7 promoter. The sequence verified plasmid was transformed into BL21(DE3)-STAR cells (Invitrogen) and single colonies were grown up overnight in Luria Broth (LB) supplemented with 50 µg/mL kanamycin. The overnight culture was used to inoculate fresh LB/kanamycin medium at a 1:1000 dilution and the culture grown at 37 °C to an OD₆₀₀ = 0.4–0.6 before induction with 1 mM IPTG and growth at 37 °C for 2–4 hours. 200 µL of the resultant culture was centrifuged, decanted, and resuspended in 2 mL of M9 minimal salts medium supplemented with 50 µg/mL kanamycin, 5 mM MgSO₄, and 1 mM IPTG. 200 µL of the resuspended culture was transferred to 96-well poly-D-lysine coated glass bottom plates (MatTek) and incubated at 37 °C for one hour. The media was then removed and the wells washed with M9/kanamycin/1 mM IPTG medium before adding 200 µL of M9 media, 1 mM IPTG, and 400 µM DFHBI-1T (Lucerna). The live fluorescence images were taken with an Andor iXon3 897 EMCCD using a 60× oil objective, an excitation filter 472/30, dichroic mirror 490 (long pass) and emission filter 520/40 on a Nikon Ti-E microscope and analyzed with FIJI⁵⁹ .