2.3 Culture conditions
The frozen stocks (100 µL) were inoculated into 50 mL LB broth with 50
mg/L ampicillin and 35 mg/L chloramphenicol at 37°C for 9 h as a seed
culture. To evaluate the effects of yeast extract supplementation on the
yields of cells and GFP, 5.0 g/L yeast extract was added to M9 minimal
salt medium composed of 12.0 g/L
Na2HPO6, 6.0 g/L
KH2PO4, 1.0 g/L NaCl, 2.0 g/L
NH4Cl, 0.5 g/L
MgSO4⋅7H2O, 4.0 g/L glucose, 30 mg/L
CaCl2⋅H2O, 20 mg/L thiamin
hydrochloride, 50 mg/L ampicillin, and 35 mg/L chloramphenicol. The seed
culture (1 mL, OD660 of approximately 5) was transferred
into 50 mL of media in a 500 mL baffled Erlenmeyer flask and incubated
at 37°C for 12 h at 200 rpm in an orbital shaker (G⋅BR-200, Taitec Co.
Ltd., Tokyo, Japan). Three hours after inoculation, 1 mM IPTG was added
to induce GFP expression. Cell growth was monitored by measuring the
turbidity at 660 nm using a spectrophotometer (V-630, JASCO Corporation,
Tokyo, Japan). GFP expression levels were measured by a
spectrofluorometer with a doubled monochrometer and a micro drop sample
holder (FP-8300, JASCO Corporation, Tokyo, Japan). For GFP
quantification, the excitation and detection wavelengths were set at 487
and 509 nm, respectively. The fluorescence intensities at these
wavelengths were used to represent GFP yields. Five microliters of
diluted culture broth were measured using spectrofluoroscopy. The
measurements were performed in at least triplicate after sampling at 0,
3, 6, 9, and 12 h.