Evaluating Mutation Rates in Synechococcus

For pre-cultivation, Synechococcus cells were inoculated in BG11 medium at an initial OD₇₃₀ of 0.2–0.3 in column photobioreactors and cultivated under 150 μmol photons/m²/s white fluorescent light at 30 °C, bubbled with air for 3 d. The pre-cultivation broth was re-inoculated into fresh BG11 medium and cultivated under the same conditions until the mid-exponential phase. Especially for MMS-treating cells, before evaluation, they were inoculated in BG11 medium at an initial OD₇₃₀ of 0.2 in a flask under 50 μmol photons/m²/s white fluorescent light at 30 °C or in column photobioreactors and cultivated in MC1000 under 42 °C and 800 μmol photons/m²/s to calculate the non-lethal mutation rates. The cell numbers were calculated using an automated cell counter (Countstar, Shanghai, China), and approximately 1 × 10⁹–4 × 10⁹ cells were collected and plated onto a solid BG11 medium containing 15 μg/mL rifampicin. The rifampin-resistant colonies were counted after 2 weeks of cultivation (ImageJ 1.52a software was used to count the rifampicin-tolerant colonies), and the frequencies of the resistant cells in the original culture broth were calculated to evaluate the mutation rates of Synechococcus genome replication. The relative mutation rate of the recombinant strains was calculated by comparing it with that of the WT. For the hypermutation evolution process, cultivation was performed in MC1000 with the temperature and light intensity set as required.