pHYαCGTd4

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

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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