pAU5-amyE

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Plasmids pAU5 and pAU5-amyE were used to transform C. glutamicum ATCC14067, resulting in ATCC14067/pAU-5 and ATCC14067/pAU5-amyE, respectively. The seed culture of the C. glutamicum strain was prepared by overnight cultivation and was then transferred to 500 mL flasks containing 100 mL of the medium for shake-culturing to produce AmyE protein.	The cell pellets and culture supernatants of the ATCC14067/pAU-5 and ATCC14067/pAU5-amyE cultures were separated by centrifugation at 13680 ×g for 5 min, respectively. The samples of the culture’s supernatants were directly used to test the production of AmyE by SDS-PAGE, Western blotting, and amylase activity analysis.

Protocol tips
Plasmids pAU5 and pAU5-amyE were used to transform C. glutamicum ATCC14067, resulting in ATCC14067/pAU-5 and ATCC14067/pAU5-amyE, respectively. The seed culture of the C. glutamicum strain was prepared by overnight cultivation and was then transferred to 500 mL flasks containing 100 mL of the medium for shake-culturing to produce AmyE protein.
Downstream tips
The cell pellets and culture supernatants of the ATCC14067/pAU-5 and ATCC14067/pAU5-amyE cultures were separated by centrifugation at 13680 ×g for 5 min, respectively. The samples of the culture’s supernatants were directly used to test the production of AmyE by SDS-PAGE, Western blotting, and amylase activity analysis.

Publication protocol

Plasmids pAU5 and pAU5-amyE were used to transform C. glutamicum ATCC14067, resulting in ATCC14067/pAU-5 and ATCC14067/pAU5-amyE, respectively. The seed culture of the C. glutamicum strain was prepared by overnight cultivation and was then transferred to 500 mL flasks containing 100 mL of the medium for shake-culturing to produce AmyE protein. The cell pellets and culture supernatants of the ATCC14067/pAU-5 and ATCC14067/pAU5-amyE cultures were separated by centrifugation at 13680 ×g for 5 min, respectively. The samples of the culture’s supernatants were directly used to test the production of AmyE by SDS-PAGE, Western blotting, and amylase activity analysis. The cell pellets were washed and re-suspended in the phosphate buffer (pH 6.0) in accordance to 1:20 (v:v) ratio of the buffer: culture medium. The cell suspension was mixed with a quarter volume of 5X SDS-PAGE loading buffer, boiled for 10 min, and centrifuged in order to remove debris, generating a sample of cell lysate for SDS-PAGE and Western blotting. The re-suspended cells were also disrupted by ultrasonication and centrifuged, generating the sample of cell homogenate supernatant for the amylase activity assay.

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