pIVEX-GAA-omega-PHACTR1-H

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	N. benthamiana plants were cultivated, infiltrated with A. tumefaciens and subsequently incubated as described before using either vacuum infiltration [39] or manual injection into leaves [27]. Whole plants or leaf discs were infiltrated with a bacterial suspension (OD600nm = 1.0) using infiltration buffer (0.5 g L−1 Fertilizer MEGA 2 (Planta Düngemittel GmbH, Regenstauf, Germany), 200 μM acetosyringone, pH 5.6). The infiltration buffer for PCPs additionally contained 50 g L−1 sucrose and 2 g L−1 glucose monohydrate. PCPs were generated from 300 μL of concentrated (200 g wet biomass L−1) continuously cultured BY-2 cells [40] and incubated in 96-well Agroprep Advance PP/PE 30–40 μm filter plates (Pall GmbH, Dreieich, Germany).

Protocol tips

N. benthamiana plants were cultivated, infiltrated with A. tumefaciens and subsequently incubated as described before using either vacuum infiltration [39] or manual injection into leaves [27]. Whole plants or leaf discs were infiltrated with a bacterial suspension (OD600nm = 1.0) using infiltration buffer (0.5 g L−1 Fertilizer MEGA 2 (Planta Düngemittel GmbH, Regenstauf, Germany), 200 μM acetosyringone, pH 5.6). The infiltration buffer for PCPs additionally contained 50 g L−1 sucrose and 2 g L−1 glucose monohydrate. PCPs were generated from 300 μL of concentrated (200 g wet biomass L−1) continuously cultured BY-2 cells [40] and incubated in 96-well Agroprep Advance PP/PE 30–40 μm filter plates (Pall GmbH, Dreieich, Germany).

Publication protocol

N. benthamiana plants were cultivated, infiltrated with A. tumefaciens and subsequently incubated as described before using either vacuum infiltration [39] or manual injection into leaves [27]. Whole plants or leaf discs were infiltrated with a bacterial suspension (OD600nm = 1.0) using infiltration buffer (0.5 g L−1 Fertilizer MEGA 2 (Planta Düngemittel GmbH, Regenstauf, Germany), 200 μM acetosyringone, pH 5.6). The infiltration buffer for PCPs additionally contained 50 g L−1 sucrose and 2 g L−1 glucose monohydrate. PCPs were generated from 300 μL of concentrated (200 g wet biomass L−1) continuously cultured BY-2 cells [40] and incubated in 96-well Agroprep Advance PP/PE 30–40 μm filter plates (Pall GmbH, Dreieich, Germany). BYL was prepared from continuously cultured BY-2 cells as previously reported [24] and used for cell-free protein synthesis. In brief, BY-2 cells were harvested during the exponential growth phase of a continuous fermentation, protoplastized and subsequently evacuolated in a Percoll gradient. In the presence of a protease inhibitor mix, the protoplasts were homogenized on ice and remaining intact nuclei and cells removed by centrifugation. Then, calcium chloride and nuclease S7 were added and the nuclease was subsequently inactivated by adding EGTA. Vector pIVEX-GAA-omega-PHACTR1-H was added to 50 μL of the lysate (BYL) in a final concentration of 80 μg mL−1 and the lysate was supplemented with creatine phosphate, triphosphate nucleotides and T7 RNA polymerase.

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