Db wax column

Extraction of fatty acids was performed as previously described [30 (link)]. Firstly, 2 g of frozen tissue powder was mixed with 15 ml of n-hexane:isopropanol (3:2, v/v) and 7.5 ml of 6.7% Na₂SO₄, followed by centrifugation for 10 minutes. The supernatant was evaporated to dryness with nitrogen. Methanol:toluene:H₂SO₄ (88:10:2, v/v/v) was added to produce fatty acid methyl esters (FAMEs). After cooling, 1 ml of heptane with 0.5 g anhydrous Na₂SO₄ was added for FAME extraction. To detect fatty acids, an Agilent 6890 N gas chromatograph equipped with a flame ionization detector and a DB-WAX column (0.25 mm, 30 m, 0.25  m; J & W Scientific) was used. The injector and detector temperatures were 230°C. The initial oven temperature was 50°C, increased to 200°C at 25°C min⁻¹, then increased to 230°C at 3°C min⁻¹. Nitrogen was used as the carrier gas at 1 ml min⁻¹. Exogenous heptadecanoic acid was added as internal standard.

Enzyme assays were carried out using previously published protocols (Ringer et al. 2003 (link), 2005 (link); Davis et al. 2005 (link)). Substrates and products for the assays were part of our chemical library. Kinetic assays of PulR were performed with 25 µg of purified, recombinant protein in phosphate-citrate buffer (pH 6), in the presence of 1 mM NADPH and with varying substrate concentration. The total volume of the assay was 200 µl. To speed up the detection of substrates and products, conditions for GC-FID (6890N, Agilent Technologies) using a DB-WAX column (60 m × 0.25 mm × 0.25 µm; J&W Scientific) were modified as follows: front inlet and detector temperature 270°C, inlet mode splitless, injection volume 1 µl, carrier gas flow (He) 0.9 ml/min; initial oven temperature 85°C (hold for 4 min), then linear gradient to 130°C at 30°C/min (hold for 3 min), and a second linear gradient to 235°C at 20°C/min (hold for 10 min). Quantitation was achieved in the ChemStation B.03.02 software (Agilent Technologies) based upon calibration curves with known amounts of authentic standards and normalization to the sample weight and peak area of the internal standard (camphor). Prism 8 software (GraphPad) was used to calculate kinetic values from assay data.

Culture volumes corresponding to 50 mg biomass were sampled on ice, centrifuged (10,000 g, 10 min at 4°C), washed twice, resuspended in 5 ml ice-cold water and stored at -20°C. Lipid extraction was performed as described previously 85 (link). Aliquots of 0.15 mL were added to 15 mL tubes and 1.5 mL of a mixture of concentrated HCl and 1-propanol (1:4) and 1.5 mL of dichlorethane were added. 400 μg of myristic acid (a 15:0 fatty acid) was included as internal standard. Samples were incubated at 100°C for 2 h. Subsequently, 3 mL of water was added to cooled samples. 1 mL of the organic phase was filtered over water-free sodium sulfate into GC vials. The fatty acid propyl esters in the organic phase were analyzed by gas chromatography (model 6890N, Agilent, U.S.A.) using a DB-wax column (length, 30 m; inside diameter, 0.25 mm; film thickness, 0.25 μm; J&W Scientific, Folsom, CA) and helium as the carrier gas. The sample volume was 1 μL, and the split was set to 1:20. The injection temperature was 230°C, and the following temperature gradient was used: 120°C at the start, increasing at a rate of 10°C/min up to 240°C, and then 240°C for 8 min. The fatty acid propyl esters were detected using a flame ionization detector at 250°C.