Experimental Design: In order to produce the CBDA synthase in the chassis organism, tobacco, several steps need to be taken. The plan for the project is 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. The first enzyme that will be introduced to the tobacco plants is Horse Radish Peroxidase(HRP), the introduction of the HRP into the tobacco will serve as a proof of concept. If the HRP can be successfully be transformed into the tobacco plants, then it is possible to introduce CBDA synthase because CBDA synthase is an enzyme similar to HRP. is A. tumefaciens will carry the genetic code for the synthesis of CBDA synthase in the hairy roots; the plants will then be feed the precursor CBGA in a bioreactor system. Upon, the feeding of the precursor the plants will excrete CBD into solution.
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 25°C. The bacteria culture was pelleted by spinning at 5,000×g for 10 minutes. The supernatant was poured off, and the pellet resuspended in nitrogen-free plant nutrient medium until the final OD600 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 25°C 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.
Inoculate one single colony of Agrobacterium in 5 ml LB with kanamycin. Grow four days at 30C. Take 1 ml of the overnight culture to inoculate 25 ml LB with kanamycin, plus 20 μM acetosyringone added after autoclaving and immediately before use, and grow overnight. Measure the of overnight culture. Precipitate the bacteria at 4,000 x g for10 minutes. Resuspend the pellet in Resuspension Solution. The final should be adjusted to 0.4. The pellet is left on the bench at room temperature overnight before infiltration. The infiltration was performed with a 5 ml syringe. The syringe (no needle) was pressed on the underside of the leaf, noting to avoid the cotyledons, and counter-pressure is exerted with a finger on the other side. Successful infiltration is often observed as a spreading “wetting” area in the leaf. The plant is checked for GFP fluorescence by a portable long-wavelength UV lamp 2-5 days after infiltration.