Team:Georgia State/Demonstrate
Demonstrate
In order to prove that CBDA synthase could indeed be transformed into tobacco plants, the enzyme horseradish peroxidase was chosen as a proof of concept. The horseradish peroxidase was ligated into the pORE vector. The pORE vector was then transformed into Agrobacterium tumefaciens. To get the A. tumefaciens into the tobacco plant, two methods of transfection were chosen: Leaf Infiltration and Hairy Root Transformation. Hairy Root Transformation The A. tumefaciens containing the binary pORE vector was grown in YEP medium for 16 hours at 25C. The bacteria culture was pelleted by spinning at for 10 minutes. The supernatant was poured off, and the pellet resuspended in nitrogen-free plant nutrient medium until the final value of 0.2 was achieved. Rock wool plugs, about 0.5 inches thick, were inoculated with 5mL of solution containing the resuspended pellet of A. tumefaciens was added to a hole in the wool. Now into the hole made in the wool a 3 cm stem from the sterile tobacco plants was placed, and the wool placed into petri dishes which were then placed into a clear storage container. The container is then covered with a lid and the container sat under ambient light at 25C for 5 days under ambient light in the flow hood. After dehydration treatment, the plugs are saturated with deionized water, and the cover of the container replaced. The plugs were then checked periodically and watered when necessary for the remainder of the induction period. After two to three weeks’ hairy roots will grow out of the rock wool. The hairy roots contain the horseradish peroxidase gene. Chemical Assay to Test Hairy Root Transformation To detect the horseradish peroxidase in the newly formed hairy roots, a chemical assay was performed. Into 5mL of deionized water(a piece of transformed hairy root along with 5mL of the chemical 3,3′,5,5′-Tetramethylbenzidine (TMB) were placed into a 15mL microcentrifuge tube. As a control, two other reactions were set up. A tube containing 5 mL of TMB with 5 mL of and a tube containing 5 mL of , 5mL of TMB, and a piece of untransformed root. The horseradish peroxidase will turn a pale blue color that can be read spectrophotometrically at 370 or 620-650nm (Sigma-Aldrich Co.LLC, n.d.)
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