Part:BBa_K1800002:Experience

HRP :Proof of Concept

In order to produce the CBDA synthase in the chassis organism, tobacco, several steps need to be taken. The first step in was to grow sterile hydroponic tobacco plants. From these sterilized plants, young sprouts will be harvested, suspended in solution, and transformed by Agrobacterium tumefaciens to produce hairy roots which contain the chosen foreign enzymes, in our case that enzyme is HRP.

Hairy Root Culture

In 2016, A. tumefaciens containing the BBa_ K1800002 in the pORE vector was grown in YEP medium for 16 hours at 25 Celsius. 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 OD 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 25 Celsuis 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 grew out of the rock wool. The hairy roots contain the horseradish peroxidase gene. See the figure below.

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. TMB is a sensitive substrate that in the presence of HRP turns a pale blue in color and can be read spectrophotometrically at 620-650 nmAs 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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