The overall goal of this procedure is to quantitatively detect protein-protein interactions in a transient expression system. This measure can help answer key questions in quan-signal transaction fields such as how to quantitatively monitor our protein protein directions after a chemical or environmental treatment. The main advantage of this technic is that it uses a luminometer or a CCD camera to detect the luciferous activity, which represents the protein protein direction intensity so the results are more accurate.
To begin this procedure, add one milliliter of a solution containing five percent sodium hypochlorite and zero point one percent triton x one hundred to a one point five milliliter microcentrifuge tube containing seeds. Let the solution rest for five minutes to sterilize the seeds. Then, wash the seeds with sterile water five times.
Suspend the washed seeds in two hundred microliters of steril water. Using fine tips, carefully place individual seeds onto the surface of plated MS agar medium. Store the plates at four degrees Celsius for three days to synchronize germination.
After this, keep medium plates under normal growth conditions for ten days. Transfer the germinating seedlings into soil, making sure not to damage the roots. Cover the tray with a transparent plastic cover overnight to maintain humidity.
Grow plants in a controlled growth room for seven weeks, when they'll be ready for agrobacterium tumefaciens infiltration. To begin, deliver one hundred and fifty nanograms of plasmids into the a-tumefaciens competent cell strain GV3101. Place the tubes containing the a-tumefaciens culture in a shaker, and incubate for four hours.
Using a glass rod, transfer the culture from the tubes to LB mediums. Culture a-tumefaciens and LB agar medium at 28 degrees celsius for four days. Next, prepare to inoculate a single a-tumefaciens colony from each plate into its own tube containing three milliliters of LB liquid medium.
Shake at 280 RBM at 28 degrees Celsius for 24 hours to propagate the culture. Then, transfer 75 microliters of the bacterial culture into 15 milliliters of fresh LB liquid medium containing 15 micrograms per milliliter of kanamycin and 50 micrograms per milliliter of rifampicin. Culture the bacteria at 220 RPM at 30 degrees Celsius for approximately 8 hours until OD600 is between 0.5 to 1.
After this, transfer the culture to 15 milliliters centrifuge tubes. Centrifuge at 4000 times gravity and four degrees Celsius for 15 minutes. Discard the supernatant and resuspend the pallet in 15 milliliters of transformation solution.
Centrifuge at 4000 times gravity and 4 degrees Celsius for 15 minutes. Then, repeat the suspension and centrifugation step once more. Discard the supernatant and resuspend the pallet in 5 milliliters of transformation solution.
Let the resuspended pallet rest for two hours at room temperature in dark conditions. Then, either add transformation solution into the OD600 is 0.5 or combine two samples with equal volume for testing paired protein interactions. Using a one milliliter needle of syringe, infiltrate suspended cells into the fourth to seventh leaves.
After this, immediately cover the plants with a black plastic cover for 12 hours. Firstly, plants should be very healthy to ensure success. Secondly, make sure not to damage any leaves during the infiltration process.
Keep the tray in darkness for 12 hours. Then grow the plants under normal conditions for 2 to 4 days before detecting the luciferase activity. To begin the imaging method, detach an infiltrated leaf.
Place the entire leaflet on a plate of MS agar medium. Using a 50 milliliter spray bottle, spray luciferon working buffer onto the leafs adaxial side. Then, keep in the dark for 5 minutes to quench the chlorophyll autofluorescence.
Use a lower light cooled CCD imagine apparatus cooled to negative 30 degrees Celsius to capture images with a light filled exposure time of 50 milliseconds, and luminescence detection time of 1 minute. After this, cut out leaf fragments from the infiltration area of the N-benthamiana leaves. Immerse the fragments in 100 microliters of deionized water in the wells of 96-well white plate.
Add ten microliters of luciferon working buffer. Let the plate rest 5 minutes. Then, perform any desired specific treatment to study protein interaction dynamics, and measure the luminescence directly with a commercial luminometer.
Shown here is the demonstration of luciferous activity to represent of COP 1 SPA 1 interactions. The luciferous activity, which is detected by luminescence, is imaged by CCD camera. COP 1 SPA 1 interactions are seen to result in copper mutation of functional luciferase.
The luciferase activity under Jasmine 8 treatment over time is then determined. COP 1 SPA 1 interactions represented by luciferase activity are seen to gradually decline in darkness. Treatment with Jasmine 8 further reduces these interactions.
Once mastered, this technic can be done in 1 week, after the plants are ready if it's performed properly. While attempting this procedure, it is important to remember to maintain healthy plants. Infiltrate leaves carefully and include controls properly.
After it's development, this technic paves the way for researchers in the field of signal transactions to explore protein protein interaction dynamics in a rapid way. After watching this video, you should have a good understanding of how to grow tobacco plants, infiltrate agrobacterias into tobacco leaves, and to detect luciferous activities.
