Chemidoc

KaiC phosphorylation in each sample was resolved by SDS-PAGE analysis using 10% Criterion Tris-HCl gels (Bio-Rad) run for 3 h at 125 V. The gels were stained with SYPRO Ruby (Invitrogen) and imaged using ChemiDoc (Bio-Rad). The ratio of phosphorylated KaiC to unphosphorylated KaiC was quantified by gel densitometry.

All M9 minimal media combinations were prepared following the base recipe²⁰ . Each treatment was supplemented with glucose to a final concentration of 0.5% (w/v). Where appropriate, additional carbon sources (glycerol, glucose, lactose, and galactose) were also added to a final concentration of 1% (w/v); for the positive control, IPTG was supplemented to a final concentration of 0.1 mM. Plates meant for BL21(DE3) additionally contained carbenicillin at a final concentration of 100 µg mL^− 1, and plates meant for SHuffle^® T7 Express (New England Biolabs C3029J) contained carbenicillin and streptomycin at final concentrations of 100 µg mL^− 1 and 25 µg mL^− 1, respectively. Starter cultures of SHuffle^® T7 Express and BL21(DE3) E. coli were prepared by inoculating eGFP transformants into 30 mL M9 medium (0.5% w/v glucose) containing the appropriate antibiotics described above. Flasks were incubated in rotary shakers set to 250 rpm at 30–37 °C for SHuffle^® T7 Express or BL21(DE3) E. coli, respectively. The BL21(DE3) culture was grown for 24 h under these conditions, whereas SHuffle^® T7 Express needed 72 h to achieve the same qualitative density. Following this initial growth, cell suspensions were plated in parallel on six M9 modifications with the appropriate antibiotic selection: M9 (0.5% w/v glucose), M9 + glucose (1% w/v), M9 + glycerol (1% w/v), M9 + lactose (1% w/v), M9 + galactose (1% w/v), and M9 + IPTG (0.1 mM). For each plate, sixteen 10 µL aliquots of homogenous culture were dotted in a 4 × 4 grid and allowed to dry under a laminar flow hood. All plates were incubated at 30 °C and were imaged after 6 days (SHuffle T7 Express) or 8 days (BL21(DE3)) of culture using a BioRad ChemiDoc™ MP imaging system. To assess in-colony eGFP fluorescence, images of each plate were captured using a 530/28 filter under UV illumination using the same exposure settings and image area. Images were analyzed using the companion ImageLab (v5.1) software, performing automatic background subtraction and quantifying integrated colony fluorescence intensity for each colony without normalization for colony size. Non-uniform or fused colonies were excluded from the analysis, resulting in 12–16 values per condition. Average fluorescence intensity was calculated for each condition.