Unless otherwise stated, all materials and reagents were of analytical grade or higher supplied by Sinopharm Chemical Reagent (Shanghai, China). Isopropyl β-d -1-thiogalactopyranoside (IPTG) and ampicillin sodium were supplied by Sangon Biological Engineering Technology and Services (Shanghai, China). Electrophoresis reagents were obtained from Bio-Rad (Hercules, CA, USA). Escherichia coli BL21 (DE3) and DH5α were obtained from Promega (Madison, WI). The Ni2+-chelating Sepharose Fast Flow resin was purchased from GE Healthcare (Uppsala, Sweden). Epilactose and lactulose were of the highest grade and were obtained from Sigma (St Louis, MO, USA). Cheese whey powder was purchased from Apple Foods Tech (Shanghai, China).
Escherichia coli bl21 de3
Manufactured by Promega
Sourced in United States
Escherichia coli BL21 (DE3) is a common bacterial strain used in molecular biology and biochemistry laboratories. It is an E. coli host strain optimized for recombinant protein expression. The strain carries the DE3 lysogen, which contains the T7 RNA polymerase gene under the control of the lacUV5 promoter, allowing for inducible expression of target proteins.
Automatically generated - may contain errors
2 protocols using escherichia coli bl21 de3
1
Enzymatic Synthesis of Galactooligosaccharides
2
Overexpression and Purification of EtSIR2A
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The PCR product of EtSIR2A was digested with the restriction endonucleases EcoRI and XhoI, and cloned into the prokaryotic expression vector pET-28a (+) (Novagen, Merck KGaA, Darmstadt, Germany). The recombinant plasmid was confirmed by DNA sequencing and transformed into Escherichia coli BL21 (DE3) (Promega, Madison, USA). Bacteria with the recombinant pET-28a (+) plasmid were induced with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG; Sigma, Louis, USA) at 37 °C for 6 h, collected by centrifugation at 10,000× g for 15 min, and sonicated on ice. The supernatant was collected, and recombinant protein was purified using a His Bind Purification kit (Novagen). Purified protein lysate was analyzed by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and its concentration was determined using a BCA protein assay kit (Novagen). The purified protein was stored in aliquots at -20 °C until further use.
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