Synchronizing Cyanobacterial Circadian Rhythms

Cells grown as described above were transferred to M9 minimal medium with no carbon source for 1 hour to synchronize the population (29 (link)). Cells were back diluted to an OD₆₀₀ (optical density at 600 nm) of ~0.1 in either 500 ml (for KaiC phosphorylation analysis) or 200 μl of M9 minimal medium (for plate reader analysis) with 0.5% succinate and 1 mM leucine to promote slower growth and without induction to reduce population desynchronization. For analysis of KaiC phosphorylation, aliquots were taken at 4-hour intervals and lysed by incubation in 3% SDS for 10 min at 100°C and visualized on a Western blot as described above. Mean normalization was performed on each time course trace from the three biological replicates. The mean-normalized, average proportion of KaiC phosphorylation was plotted. For fluorescence time courses of the synthetic reporter, mineral oil was added to prevent evaporation. Readings were taken using a Synergy NEO HTS MultiMode microplate reader (BioTek) using appropriate filters. Fluorescence readings were normalized to OD₆₀₀, and background (OD₆₀₀ normalized fluorescence readings from a reporter-only strain AHC157) was subtracted. For both KaiC phosphorylation and synthetic reporter time courses, statistical analyses were performed with RAIN (7 (link)) using a periodicity of 24.8 hours over a 3-day time course.