Shim pack vp ods column

For ATPase measurements, KaiC proteins fused to an N-terminal Strep-tag were used, and all Kai proteins were purified via size exclusion chromatography (described above). Strep-KaiC3 or Strep-KaiC3 variants at a concentration of 3.45 μM protein were incubated in ATPase buffer (20 mM Tris-HCl [pH 8], 150 mM NaCl, 1 mM ATP, 5 mM MgCl₂) at 25°C, 30°C, or 35°C for 24 h. To analyze the influence of KaiA₆₈₀₃ and KaiB proteins on KaiC3 ATPase activity, we mixed 0.2 mg ml⁻¹ KaiC3 with 0.04 mg ml⁻¹ KaiA₆₈₀₃ or 0.04 mg ml⁻¹ KaiB and incubated the mixtures for 24 h at 30°C. Monomeric and oligomeric KaiB3 were analyzed separately. To monitor ADP production, every 3, 4, or 6 h, 2 μl of the reaction mixture was applied on a Shim-Pack-VP-ODS column (Shimadzu) and separated using 100 mM phosphoric acid, 150 mM triethylamine, 1% acetonitrile as running buffer. ADP production per monomer KaiC and 24 h was calculated using a calibration curve. A detailed protocol can be found on protocols.io (https://doi.org/10.17504/protocols.io.mebc3an). The Q₁₀ value was calculated from ATPase measurements at 25°C and 35°C, using the formula $Q_{10}={\left(\frac{R_2}{R_1}\right)}^{10°\text{C}/(T_2-T_1)}$ .

Stevia leaves for SGs content analysis were harvested from the sixth node of plants grown in the greenhouse for 3 weeks. After drying the leaves overnight in a 60 °C oven, the samples were ground and 30 mg of the powdered leaves were extracted using 3 mL of water twice in an ultrasonic bath maintained at 50 °C for 20 min. The extracts were centrifuged at 1800 × g for 15 min. One microlitre of supernatant filtered through a 0.45 μm filter was loaded onto a solid phase extraction (SPE) column C2 (Agilent, Santa Clara, CA) and washed with acetonitrile:water (20:80, v/v) before elution in 1 mL of methanol:acetonitrile (50:50, v/v). To analyse SGs content, 5 μL of the eluted sample was applied on a Shimadzu Nexera ×2 ultra‐high performance liquid chromatography (UHPLC) fitted with a Shim‐pack VP‐ODS column (250 × 4.6 mm, i.d. 5 μm) and detected by a photodiode array detector (SPD‐M30A with high sensitivity cell). The elution was performed over 24 min with a 30%–80% acetonitrile gradient at a flow rate of 1.0 mL/min. Column oven was maintained at 40 °C. Chromatogram detected at a wavelength of 210 nm was used for SGs identification and quantification. Peak assignment was based on comparison with elution profile of known standards (ChromaDex,Irvine, CA) and the concentration of each SG was determined from the standard curves of the respective SGs.

HPLC/HRMS measurement was conducted on the Ailent 6530 Accurate-Mass Quadrupole Time-of-Flight (Q-TOF) equipped with Agilent 1260 HPLC in a full scan in positive mode (ESI+). During HPLC analysis, purified compound X was detected at 254 nm and eluted with a 40 min gradient as described as above (section: Metabolite analysis of streptomycete cultures) through a Shim-pack VP-ODS column (5 μm, 250 L×4.6 mm, Shimadzu). During subsequent HRMS analysis, the parent ion with m/z 301.0713 for compound X (C₁₆H₁₃O₆ ([M+H]⁺)) at ca.21.8 min retention time was monitored.