pHYαCGTd4

Protein Expression Prokaryotic cells - B. subtilis Pullulanase

Experiment
Protein Expression Prokaryotic cells - B. subtilis Pullulanase
Product
pHYαCGTd4 from Jing Wu, State Key Laboratory of Food Science and Technology, Ji
Manufacturer
Jing Wu, State Key Laboratory of Food Science and Technology, Ji

Protocol tips

Protocol tips
For protein expression, the plasmids constructed were used to transform B. subtilis CCTCC M 2016536 competent cells using the method of Anagnostopoulos and Spizizen.

Publication protocol

For routine plasmid construction, E. coli JM109 was incubated in LB medium supplemented with 100 mg/L ampicillin for 10 h at 37 °C with shaking at 200 rpm. To express β-CGTase in shake-flask fermentations, B. subtilis CCTCC M 2016536 plasmid strains transformed with the appropriate plasmids were incubated in 10 mL of LB medium supplemented with 20 mg/L tetracycline for 10 h at 37 °C with shaking at 200 rpm. A portion (2.5 mL; 5% [v/v]) of this overnight culture was used to inoculate 50 mL of TB medium containing 20 mg/L tetracycline, which was then incubated for 48 h at 30 °C with shaking at 200 rpm. The culture was harvested by centrifugation at 12,000×g for 10 min at 4 °C to obtain the culture supernatant, which contained reporter proteins.

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