pGFPe-araHWT

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	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.

Protocol tips

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.

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).

"

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Protein Expression Prokaryotic cells - E. coli araHWT using pGFPe-araHWT from Daniel O. Daley, Center for Biomembrane Research, Department of .

Manufacturer protocol

Download the product protocol from Daniel O. Daley, Center for Biomembrane Research, Department of for pGFPe-araHWT below.

Videos

Check out videos that might be relevant for performing Protein Expression Prokaryotic cells - E. coli araHWT using pGFPe-araHWT from Daniel O. Daley, Center for Biomembrane Research, Department of . Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.