Team:British Columbia/Notebook/Protocols/GCMS

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Gas chromatography-mass spectrometry for biosynthetic product detection

Clones are grown in LB and chloamphenicol (15 µg/mL) overnight. Cultures are then diluted 1/100 in M9 minimal media with 2% glucose containing 2 mM of substrate of interest. If the construct is being assessed under induction, 10 µg/mL of arabinose was added at this point.

Cultures are incubated at 37 °C for 7 hours before partially lysis by the addition of 1% of Triton-X for 20 min. In the case where bioconversion is needed by in vitro conversion (by co-culturing strains harboring sequential steps in a pathway), the lysed cells are incubated for another 3 hours at 37 °C.

2 mL of the minimal media cultures are then centrifuged at 10,000 x g for 30 minutes.

The supernatant is then transferred to a glass tube, and extracted twice with equal volumes of ethyl acetate (using glass pipettes).

Samples are then dried under nitrogen before resuspension in 50 µL of pyrimidine.

Samples are then derivatized using BSTFA+TMCS- (99:1). GC–MS is performed using an HP 6890 series GC system fitted with an HP 5973 mass-selective detector and a 30 m × 250 μm HP-5MS Agilent column.

The operating conditions were TGC (injector), 280 °C; TMS (ion source), 230 °C; oven time program (T0 min), 120 °C; T2 min, 120 °C; T30 min, 260 °C (heating rate 5 °C min^–1); and T37 min, 260 °C.