Nicotianabenthamianaagroinfiltration
-Transfer the GBelements listed as ‘GBelements used in the experiment’ to Agrobacterium tumefaciens strain GV3101 by electroporation.
-Set overnight-grown bacterial cultures for agroinfiltration.
-Pellet and resuspend the cultures on agroinfiltration solution (10mM MES, pH 5.6, 10mM MgCl2, and 200µM acetosyringone) to an optical density of 0.2 at 600nm.
-Incubatethe bacterial suspensions for 2 hours at room temperature on a horizontal rolling mixer.
-Mix the bacterial suspensions in equal volumes.
-Agroinfiltratethe mix on the three youngest leaves of each plant through the abaxial using a 1ml needle-free syringe.
Sampling
-Four days after infiltration (more or less 96 hours), excise one disc (0.8 cm in diameter, approximately 18-19mg) of each agroinfiltratedleaf.
Luciferase/Renilla activity determination
-Homogenize leaf discs in 1.5ml eppendorfs using steel balls and the RETSCH ball mill MM 400.
-Extract each sample with 150µl of ‘Passive Lysis Buffer’.
-Vortex it andcentrifugue it 15 minutes (14000 x g) at 4ºC.
-Take the supernatant and dilute it 2:3 in Passive Lysis Buffer (24µl of supernatant + 36µl of Passive Lysis Buffer) resulting in the working plant extract.
-For Flucactivity determination use a white 96-well plate. On each well, mix10µl of working plant extract with40µl of LARII. Incubate for 10 minutes and measure the luciferase activity using a GloMax 96 MicroplateLuminometer (Promega) with a 2-second delay and a 10-second measurement.
-For Rluc activity determinationadd 40µl of Stop&Glo Reagent with the renilla substrate to each well of the plate.Incubate for 10 minutes and measure the luciferase activity using a GloMax 96 MicroplateLuminometer (Promega) with a 2-second delay and a 10-second measurement.
Data analysis
-Calculate the Fluc and Rluc mean for each well based on the three measurements.
-Calculate the Fluc/Rluc ratio for each well.
-Express the Fluc/Rluc ratios as RPUs (relative promoter units) by dividing each ratio by the Fluc/Rluc ratio obtained for the reference standard GB0166 that has to be expressed in a different plant on the same experiment.