Twin mixer tm 282

After 3 days of cultivation, Synechocystis 6803 cells (OD₇₃₀ × culture volume [mL] = 150) were collected by centrifugation (5,800 × g for 2 min at 25°C). The cells were suspended in 600 μL of 60% (vol/vol) methanol, and the suspension was mixed using a Twin Mixer TM-282 (Asone, Osaka, Japan) for 15 min. The suspension was centrifuged (20,400 × g for 5 min at 4°C), and 500 μL of the supernatant was collected. The supernatant was added to an Amicon Ultra 3-kDa-cutoff filter (Merck, Billerica, MA, USA) and centrifuged (20,400 × g for 60 min at 4°C). After centrifugation, 350 μL of the filtrate was dried using a CVE-2000 centrifugal evaporator (Eyela, Tokyo, Japan). The residue was dissolved in 30 μL of 100 mM Tris-HCl buffer (pH 8.0). The concentration of malate in the sample was measured using an E-kit for liquid l-malate (J. K. International, Tokyo, Japan).

After 3 days of cultivation, Synechocystis 6803 cells [OD₇₃₀ × culture volume (mL) = 100] were harvested by centrifugation [5800 × g for 4 min at 25 °C]. The cells were suspended in 600 μL of 60% (v/v) methanol and mixed with TWIN MIXER TM-282 (Asone, Osaka, Japan) for 15 min. The suspension was then centrifuged. A volume of 500 μL of the supernatant was transferred to an Amicon Ultra 3-kDa-cutoff filter (Merck, Billerica, MA, USA) and centrifuged. After centrifugation, 350 μL of the filtered fraction was dried using a CVE-2000 centrifugal evaporator (EYELA, Tokyo, Japan). The pellet was dissolved in 200 μL of 100 mM Tris-HCl buffer (pH 8.0). The quantification of aspartate and glutamate was performed using the Aspartate Assay Kit (MAK095) (Sigma-Aldrich, St. Louis, MO, USA) and F-kit l-glutamate (J.K. International, Tokyo, Japan).