Shake flask experiments
To prepare a stock culture, one colony of B. subtilis168/pHT01-aprN1 was inoculated in 1 mL Luria-Bertani (LB) medium
and then incubated overnight at 37°C in a shaking incubator at 200
revolutions per minute (rpm). A flask with 25 mL fed-batch culture media
was then inoculated with 7.5% (v/v) of stock culture and incubated at
37°C until reaching mid-log phase (OD600 of 0.8–1.0).
Next, the temperature was adjusted to 28°C and held for 20 min, after
which a 25-μL aliquot of 0.5 mmol L-1 isopropyl
β-D-1-thiogalactopyranoside (IPTG) was added and the culture was further
incubated for 45 h at 28°C in a shaking incubator (200 rpm) for NK
expression.