title: WGS of S. elongatus PCC 7942 CscB/SPS: oxygen stress adapted population 1
design: Synechococcus elongatus PCC 7942 CscB/SPS strain, engineered to overexpress sucrose phosphate synthase (SPS) and sucrose permease (CscB), was obtained from Dr. Daniel Ducat. Cultures were maintained in BG11 medium supplemented with chloramphenicol (12.5 mg L?) and kanamycin (25 mg L?). Standard growth conditions were maintained at 292C with nitrogen gas sparging supplemented with 2% CO? under 200 ?mol photons m? s? illumination from LED lights. Culture mixing was facilitated using PFTE Octagon Spinbar Magnetic Stirring Bars (9.5 mm x 25.4 mm) at 200 r.p.m. Antibiotics were removed prior to experimental procedures. During the experiment, continuous cultivation was performed in 6.5-L New Brunswick Bioflo 310 fermentors (Eppendorf, Inc.) equipped with custom LED photobioreactors. Steady-state cultures (5.5-L) were maintained in turbidostat mode at OD??? of 0.08, with constant conditions of 250 rpm agitation, 30C, and pH 8.0 (controlled by 2 M NaOH or HCl addition). Illumination was provided at 760 ?mol photons m? s? using 630 and 680 nm narrow-band LED illuminator chips (Marubeni America Corporation). Photosynthetically active radiation (PAR, 400-700 nm) was monitored using three quantum sensors (LI-210SA, LI-COR Biosciences). Initial conditions utilized a baseline sparging rate of 2.04 L min? with 98:2 N2/CO2 gas mixture. Oxidative stress was implemented through stepwise increases in O2 content while maintaining constant total gas flow and CO2 concentratio, with corresponding N? reduction. Dissolved O? was measured using a Clark-type electrode (InPro 6800Series, Mettler Toledo). Turbidostat operation was maintained by correlating transmitted irradiance measurements with spectrophotometer-verified OD750 values to control medium addition and removal.Adaptive evolution under progressive oxygen stress: Cultures were subjected to iterative cycles of increasing oxygen concentrations in turbidostatic growth conditions. The ancestral population was exposed to 0-78.4% oxygen cycling to 0% oxygen. Adapted population one was exposed to 0-93% oxygen cycling to 0% oxygen. Adapted population two was exposed to 0-99.8% oxygen cycling to 0% oxygen. DNA samples were collected at 0% oxygen from the ancestral strain and from each adapted population (populations 1, 2, and 3) to track accumulation of mutations during adaptive evolution. DNA was extracted using E.Z.N.A. Soil DNA Kit. Library construction and sequencing was done at Azenta Life Sciences using Non-human whole genome sequencing bundle, 2x150bp, 10 Gb per sample