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Ecological and Evolutionary implication of Bt cotton. Measurement of a single gene difference in two cotton plants by PCR

BIG IDEA Genetic Engineering has allowed agriculture to move into a new dimension of artificial selection of desirable traits for crops. Since the beginning of agriculture, humans have selected for desirable traits in their crops, these traits tend to not be good for the survival of the plants, hence artificial and not natural selection. The new ability to add and remove genes from plants, agriculture now does not need to wait for the plant to evolve the gene during random mutagenesis of its own genome. Plants might not be able to evolve some traits due to limitation of its DNA sequences. For example, we may be able to grow a tail because it is in our genetics, with a simple mutation we may grow a tail, but we do not have the genetics or the ability to evolve suction cups on the bottom of our feet, such as insects have. Never the less, Spiderman is still an amusing fantasy.
This activity will allow you to investigate the single gene difference of a genetically modified organism, Bt cotton. You will discuss the ecological and evolutionary implications of having cotton produce Bt toxin. Bt toxin is a chemical made by a gene found in a bacteria that lives in the soil, Bacillus thuriniensis. The chemical is toxic to some insects that happen to devour agricultural crops. Insertion of Bt gene into these crops has resulted in a dramatic decrease in the amount of pesticides used to grow these crops.

Let us look at two cotton plants, do they look different from each other? If so how and what implications can you make from these observations? You will analyze the DNA of these two plants and determine which one has the gene for Bt cotton. Keeping in mind what a cell does when it replicates DNA, make a list of steps that you think would be necessary for the replication of a single gene by Polymerase Chain Reaction.

Do you think that cotton could evolve the ability to produce the Bt toxin on its own? Why or why not?

DNA extraction from cotton leaf

Materials/Equipment Needed
For the class

- Heating Block
- Pipetman
- Pipet tips
- Sigma XNAP Extract N Amp for Plants Kit
- Bt and nonBt cotton
- 1.5 ml tubes
- Hole punch
- Forceps
- Vortex

1. Punch a 0.5 to 0.7 cm disk of leaf tissue into a 1.5 ml tube marked (E) using a hole paper punch. Do not use the same hole punch for different plant types. The tube has a premeasured (100 µl) amount of Extraction Solution.
2. Close the tube and vortex briefly. Make sure the disk is covered by the Extraction Solution.
3. Incubate at 95 °C for 10 minutes. Note that leaf tissues usually do not appear to be degraded after this treatment.
4. Add 100 µl of Dilution Solution (the entire contents in the tube marked (D) and vortex to mix.
5. Store the leaf extract in the refrigerator or use directly for PCR amplificaiton. It is not necessary to remove the leaf disk before storage.

PCR amplification

Materials/Equipment Needed
For the class

- Thermocycler
- Micropipet and tips
- 0.2 ml PCR microcentrifuge tube
- Forward and reverse Bt primers
- Extract-N-Amp PCR ReadyMix

For each reaction add the following reagents to a thin-walled 0.2 ml PCR microcentrifuge tube:

• 5 µl of primer mixture
• 5µl of Leaf disk extract
• 10 µl of Extract-N-Amp PCR ReadyMix
  Total volume 20 µl
  Mix gently by pipetting

Place tubes into thermocycler and select the BT1 program which has the following parameters:
- Initial Denaturation 94 °C 5 minutes
- Denaturation 94 °C 30 seconds
- Annealing 55°C 1minutes
- Extension 72 °C 1minutes
- Final Extension 72 °C 5 minutes
- Hold 4 °C 24 hours

The amplified DNA is ready for analysis by gel electrophoresis. If you cannot electrophoresis the DNA the next day, store the PCR products in the freezer.

Electrophoresis of your PCR reactions

Materials/Equipment Needed

- Electrophoresis apparatuses, electrodes, and power supplies
- Micropipet
- Micropipet tips
- Loading dye
- 0.8% agarose gel
- Molecular weight markers
- Water bath at 55°C or hot plate
-Thermometer for water bath
- TAE buffer
- Ethdium Bromide staining sheets
- Staining tray
- UVlight box
- Polaroid Camera

Procedure

Pouring an agarose gel

1. Get your electrophoresis apparatus and seal both ends of the gel tray with stoppers.

2. Make sure one comb is in place at the negative electrode (black end of the gel).

3. Pour melted agarose into the gel space until the gel is about 5 mm deep. Let the agarose harden, which should take 5-10 minutes. Don’t touch/move your gel until it’s hard. In the meantime, prepare your PCR reactions for electrophoresis.Electrophoresis of your PCR reactions

1. Using the micropipet with a clean tip, pipet 4 µl gel loading solution into your PCR reaction tube.
You will load both your PCR reactions and standard DNA markers sample into the gel. A standard DNA marker has a bunch of different sized pieces of DNA so you can compare it to the DNA from your PCR reaction to figure out what size piece it is.

3. When your gel has hardened, remove the stoppers.

4. Load all of of your PCR sample into a well, use the 20 µl pipet- be sure you keep track of which samples you're loading in which wells. Load 10 µl of DNA marker into a well.

5. Pour TAE buffer carefully so it fills the electrophoresis apparatus and just covers the gel.

6. Run that gel! Plug the electrodes into your electrophoresis apparatus (red to red, black to black),
being careful not to bump your gel too much.

7. Plug the power source into an outlet and set the voltage to about 100 V (max = 120 V).

8. Let the gel run until the dye migrates about 1/2 through the gel (about 20-25 minutes).

9. Turn off the power supply, disconnect the electrodes, and remove the top of the electrophoresis
apparatus.

Staining gels to examine PCR reactions (for teachers only, Ethidium Bromide is a mutagen!!!)

1. Place gel in staining tray
2. Moisten the top of the gel with a little buffer, then place the Ethidium Bromide sheet on top of the gel.
3. Stain for about 10-15 minutes.
4. Carefully place the gel on the UV light view your gel.
5. Photograph the gel with the polaroid.

Analysis

What do you see on your gel? Is the DNA the same in the two plants? What do you think the sequence of this DNA would be if you were to sequence it? Because a genetic change in an organism is a relatively permanent change, how do you think this would affect the development of insect resistance compared the more conventional method of spraying against insects? As with all technology in our society, there are good and bad points, as an individual in this world you need to be able to weigh the good versus the bad. Make a list of the good and bad parts of Genetically Engineered crops, and weigh the dangers versus the benefits (feel free to include food stuff in this section).

Based om what you know about random mutation of genes, speculate what will happen to a large group of insects trying to survive in the presence of plants producing Bt toxin.

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BIOTECH Project Department of Molecular and Cellular Biology The University of Arizona October 6, 1997 Last Modified March 5, 2002 Nadja Anderson, Ph.D. nadjal@email.arizona.edu http://biotech.biology.arizona.edu