Publication protocol
"Protein expression was carried out as described previously [9], with a few modifications. Expression vectors were transformed into the E. coli strain BL21(DE3) pLysS and overnight cultures were prepared by inoculating a single colony in 800 μl Luria–Bertani (LB) liquid media with 34 μg/ml chloramphenicol and 50 μg/ml kanamycin. Cultures were incubated in a 2.2 ml 96-well plate overnight at 37 °C with shaking. Overnight cultures were back-diluted 1:50 in LB plus antibiotics in a 5 ml 24-well growth plate, and grown at 37 °C with shaking to an OD600 of approximately 0.3. Synthesis of proteins was induced by addition of either 0.1, 0.5 or 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) and incubation for a further 20 h at either 25 or 37 °C. Optimum conditions for araHWT, araH5′OPT, araH5′OPT-SYN1, araH5′OPT-SYN2, araH+84WT, narKSYN1, narKΔ5′WT and nark+84WT were obtained by induction with 1 mM IPTG at 25 °C. Optimum conditions for araHSYN1, araHSYN2 and narK5′OPT-SYN2 were obtained by induction with 1 mM IPTG at 37 °C. Optimum conditions for araHΔ5′WT were obtained by induction with 0.5 mM IPTG at 25 °C, and for narKWT, narKSYN2, narK5′OPT and narK5′OPT-SYN1 by induction with 0.5 mM IPTG at 37 °C. The OD600 of the culture was measured in a Spectramax M2e (Molecular Devices). Cells were then harvested by centrifugation at 3220×g for 20 min, resuspended in buffer (50 mM Tris–HCl at pH 8.0, 200 mM NaCl, 15 mM EDTA) and transferred to a 96-well optical bottom plate. Fluorescence was read in a Spectramax Gemini (Molecular Devices) at excitation and emission wavelengths of 485 and 513 nm respectively. The amount of GFP produced (in mg/l) was calculated using a standard curve obtained from purified GFP mixed with whole cells (to account for quenching). The amount of AraH and NarK produced (in mg/l) was derived from the amount of GFP in the cell (assuming a 1:1 ratio of AraH/NarK to GFP).
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