Dfhbi 1t

(5Z)-5[(3,5-Difluoro-4-hydroxyphenyl)methylene]-3,5-dihydro-2-methyl-3-(2,2,2-trifluoroethyl)-4H-imidazol-4-one (DFHBI-1T) was purchased from Tocris Bio-techne. A DFHBI-1T stock solution (20 mM) was prepared in DMSO and stored in the dark at −20 °C for a week. All RNA oligonucleotides were purchased from Eurogentec and resuspended in ultrapure water to give stock solutions of 100 μM and stored at −80 °C. All DNA oligonucleotides and gBlocks Gene Fragment were purchased from IDT. The DNA oligonucleotides were resuspended in ultrapure water to give stock solutions of 100 μM and stored at −20 °C. The gBlocks Gene Fragment was resuspended at a final concentration of 10 ng µL⁻¹ and stored at −20 °C. EDTA 0.5 M pH 8.0, Ultra Pure^TM 10x TBE buffer, Ultra Pure^TM 1 M Tris HCl pH 7.5, 10x TAE buffer pH 8.5, Ultra Pure^TM distilled water, SYBR® Gold, 6% Novex^TM TBE gel and 10% TBE-Urea gel were purchased from Invitrogen, Life Technologies. HEPES 1 M pH 7 Bioreagent, KCl 1 M BioUltra, MgCl₂ 1 M in water BioUltra, 3-aminopropyltriethoxysilane (APTES) and agarose were purchased from Sigma-Aldrich.

Transcriptional optimization was performed separately for each species. Cell lysates were combined with amino acids, PEG, and energy buffer as previously described (Sun et al, 2013), with additional Mg‐glutamate and K‐glutamate (both from Sigma‐Aldrich) at concentrations of 0, 3, 6, or 9 mM and 0, 30, 60, or 90 mM, respectively (total 16 combinations), in a skirted white 96‐well PCR plate (Bio‐Rad). A plasmid construct (pTOPO‐F30‐Broccoli) containing strong broad‐host‐range promoter (Gen_18145) identified by our previous study (Johns et al, 2018), F30‐Broccoli, and B0015 terminator was used as a DNA template, and nuclease‐free water was used as a negative control. DNA template (2 μl) and 10 mM DFHBI‐1T (0.5 μl, Tocris Bioscience) were added to each well immediately before time‐course measurements. Fluorescence was tracked for 8 h using a Synergy H1 plate reader (BioTek) at 30°C using excitation and emission wavelengths of 482 and 505 nm, respectively. The concentrations of Mg‐glutamate and K‐glutamate yielding the highest fluorescence peak were used for future experiments, typically between 1 and 4 h.