3.4 Optimization of production conditions for POI
A convenient quantification method for the expressed POI is essential for the optimization of bioproduction conditions. The relationship between FL-DT fluorescence intensity and the concentration of N2C was then investigated by incubating 0.01-1.00 equivalent of N2C withFL-DT for 30 min (Figure S7B ). The presence of N2C triggered the release of fluorescence, which is also observed under a handheld UV lamp (Figure 4C ). The changes in fluorescence intensity were in a linear relationship with the concentrations of the added N2C (Figure S7C ). The limit of detection was determined to be 2 nM. The concentrations of N2C expressed in E. coli under variable production conditions, including the concentration of inducer isopropyl-β -D-thiogalactopyranoside (IPTG), induction time, and incubation temperature, were then quantified with FL-DT to determine the optimal parameters. Initially, three IPTG concentrations (0.1 mM, 0.5 mM, and 1.0 mM) were investigated for their impacts on protein expression. Each test group was subjected to 3 parallel experiments at 30 °C with an incubation time of 20 h. The fluorescence intensity was recorded and converted to protein concentration (method described in supplementary materials). The result reveals that the concentration of inducer IPTG barely affects the N2C production efficiency (Figure S7D ). By fixing the inducer concertation at 0.5 mM, the effect of different induction temperatures (20 °C, 25°C, 30°C, 37°C) on protein expression at 20 h was explored, showing an optimal induction temperature of 30°C (Figure 4D ). The contents of N2C expression after 8 h, 12 h, 16 h, 20 h, and 24 h were then compared under 30°C with the inducer concertation of 0.5 mM(Figure 4E ). The effect of induction temperature and culture time have a remarkable influence on the expression of recombinant protein, and the optimized condition for N2C protein production of ~0.48 mg/mL is to incubate for 20 h at 30 °C.