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.