SEC-GFP-pHis1522

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Brevibacillus competent cells were prepared according to the manufacturer’s instructions (Takara Bio Inc., Shiga, Japan).	All the expression vectors were transformed into Brevibacillus choshinensis SP3 using the tris-polyethylene glycol method [4] as described by Takara Bio manual using 0,5-2 μg of total DNA. 10 μg/mL of neomycin and 20 μg/mL of tetracycline were respectively used to transform the plasmid constricts. To screen for protein production freshly transformed cells were grown overnight in 10 mL of TM medium containing 10 μg/mL of neomycin (pNI-His and pNC-His vectors) or 20 μg/mL of tetracycline (pHis1522 and SEC-pHis1522 vectors) at 30°C, 150 rpm. Pre-inoculums were inoculated 1:100 in 50 mL of the same media and grown at 150 rpm at 25°C, 30°C and 37°C.	Recombinant expression of the selected proteins under transcriptional control of the xylose-inducible promoter, were induced by the addition of xylose at different concentrations (0,5-1-2%) at OD600 about 1,5. Intracellular GFP, α-amylase and TcdA-GT samples were withdrawn at different growth phases, and subjected to Western blotting analysis, GFP fluorescence determination and α-amylase activity assay as described below.

Upstream tips
Brevibacillus competent cells were prepared according to the manufacturer’s instructions (Takara Bio Inc., Shiga, Japan).
Protocol tips
All the expression vectors were transformed into Brevibacillus choshinensis SP3 using the tris-polyethylene glycol method [4] as described by Takara Bio manual using 0,5-2 μg of total DNA. 10 μg/mL of neomycin and 20 μg/mL of tetracycline were respectively used to transform the plasmid constricts. To screen for protein production freshly transformed cells were grown overnight in 10 mL of TM medium containing 10 μg/mL of neomycin (pNI-His and pNC-His vectors) or 20 μg/mL of tetracycline (pHis1522 and SEC-pHis1522 vectors) at 30°C, 150 rpm. Pre-inoculums were inoculated 1:100 in 50 mL of the same media and grown at 150 rpm at 25°C, 30°C and 37°C.
Downstream tips
Recombinant expression of the selected proteins under transcriptional control of the xylose-inducible promoter, were induced by the addition of xylose at different concentrations (0,5-1-2%) at OD600 about 1,5. Intracellular GFP, α-amylase and TcdA-GT samples were withdrawn at different growth phases, and subjected to Western blotting analysis, GFP fluorescence determination and α-amylase activity assay as described below.

Publication protocol

"Brevibacillus competent cells were prepared according to the manufacturer’s instructions (Takara Bio Inc., Shiga, Japan). All the expression vectors were transformed into Brevibacillus choshinensis SP3 using the tris-polyethylene glycol method [4] as described by Takara Bio manual using 0,5-2 μg of total DNA. 10 μg/mL of neomycin and 20 μg/mL of tetracycline were respectively used to transform the plasmid constricts.
To screen for protein production freshly transformed cells were grown overnight in 10 mL of TM medium containing 10 μg/mL of neomycin (pNI-His and pNC-His vectors) or 20 μg/mL of tetracycline (pHis1522 and SEC-pHis1522 vectors) at 30°C, 150 rpm. Pre-inoculums were inoculated 1:100 in 50 mL of the same media and grown at 150 rpm at 25°C, 30°C and 37°C. Recombinant expression of the selected proteins under transcriptional control of the xylose-inducible promoter, were induced by the addition of xylose at different concentrations (0,5-1-2%) at OD600 about 1,5. Intracellular GFP, α-amylase and TcdA-GT samples were withdrawn at different growth phases, and subjected to Western blotting analysis, GFP fluorescence determination and α-amylase activity assay as described below."

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Download the product protocol from Manuele Martinelli, Novartis Vaccines and Diagnostics, Via Fiore for SEC-GFP-pHis1522 below.

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