2 Creating Genetically Modified Bacteria Gl o w - i n-th e - d a r k rabbits, pigs, and mice may sound like something out

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1 2 Creating Genetically Modified Bacteria Gl o w - i n-th e - d a r k rabbits, pigs, and mice may sound like something out of a science fiction movie, but because of genetic modification, these animals actually exist. They were the results of scientists inserting a gene from the jelly species Aequorea victoria into their DNA. Genes code for the production of specific proteins. Aequorea jellies naturally glow in the dark because they have a gene that codes for green fluorescent protein. The goal of inserting a gene from one organism into another is for the modified organism to make the protein coded by the inserted gene and express the new trait. For example, a certain gene in Bacillus thuringiensis bacteria produces the Bt pesticide protein. When this gene is inserted into corn, the resulting Bt corn produces the Bt pesticide. This production of a protein is called gene expression. A gene from an Aequorea jelly (a) has been inserted into the DNA of the mouse (b), causing it to glow. a b 269

2 Science & Global Issues/Biology genetics In order to produce a genetically modified organism, scientists insert the desired gene into the DNA of eggs from the target organism. Scientists often include the gene for an Aequorea jelly s green fluorescent protein, in addition to the desired gene. If they can successfully raise these eggs to adult organisms, and if that organism glows, scientists know that they have inserted the genes correctly. The green fluorescent protein acts like a marker that shows the genes have been inserted into the target organism and that the organism can express the trait. In this activity, you will genetically modify a population of Escherichia coli (E. coli) bacteria. Geneticists study E. coli because, even though it is a simple organism, it uses the same cellular processes for gene expression as do more complex organisms. You will insert two genes into E. coli: one for green fluorescent protein (GFP), and one that will make the E. coli resistant to the antibiotic ampicillin. Because the plates on which you will grow the bacteria contain ampicillin (which normally kills E. coli), only the successfully modified E. coli will grow. Challenge 00How do scientists genetically modify an organism? Materials For the class 4 6 E. coli starter plates tube of pglo (GFP) plasmid waste container holding 10% bleach solution spray bottle of disinfectant ultraviolet (UV) light supply of paper towels For each group of four students container of crushed ice 2 microtubes containing 300 L CaCl 2 inoculating loop 4 sterile pipettes permanent marker timer 2 Luria broth (LB) ampicillin plates 2 spreaders For each student Student Sheet 2.1, Genetic Modification Procedure Student Sheet 2.2, E. coli Growth Observations Student Sheet 2.3, Genetics Case Study Comparison 3 sticky notes safety goggles 270

3 creating Genetically Modified bacteria Activity 2 Procedure 1. Follow your teacher s instructions for recording notes on this laboratory. 2. Read the entire procedure to familiarize yourself with the steps. After doing so, on Student Sheet 2.1, Genetic Modification Procedure, write a summary of the purpose of this activity and the experimental design you will follow to transform the E. coli bacteria. 3. Sterilize your table surface with disinfectant. It is important to work on sterile surfaces during this investigation so that your bacteria do not become contaminated. 4. Label one of the microtubes containing CaCl 2 1 pgfp, and label the other Control. Place both tubes in your beaker of crushed ice. 5. With the inoculating loop carefully scrape about ¼-loop-full of E. coli bacteria from the starter plate. To prevent contamination touch only the handle of the loop. Be careful not to damage the agar plate while harvesting the bacteria. 6. Place the loop with the bacteria into the CaCl 2 solution in tube 1 pgfp, and twirl it back and forth in the liquid for a few seconds to be sure the bacteria have come off the loop. Remove the loop, and place it on a clean paper towel. Do not touch the loop touch only the handle to prevent contamination. 7. Your solution should turn cloudy with E. coli. If not, use a sterile pipette to mix the cells by gently suctioning the solution into the pipette and then pushing it back out into the microtube. Repeat the mixing 4 to 5 times. Place the pipette on the paper towel with the loop. Close the tube, and put the tube back on ice. Note: Touch only the bulb end of the pipette, to avoid contamination. Be careful to only suction the solution far enough to remove it from the tube; do not let any get into the bulb of the pipette. Do this gently to prevent any bubbles from forming in the solution. Safety Be cautious when working with live organisms. If there are any spills, or if any substances come in contact with your skin, notify your teacher immediately, and wash with soap and water. Wash your hands at the end of the investigation. Do not look directly at the UV light source, as it might damage your eyes. Follow the sterile technique procedures outlined in the box on the next page. 8. Repeat Steps 5 7 for the Control tube. You may use the same loop and pipette, as long as they have been kept on a clean paper towel. 9. Obtain the pgfp plasmid according to your teacher s instructions. With a new pipette, transfer 10 µl of pgfp plasmid to the 1 pgfp tube. Close the tube. Mix the contents by flicking the tube vigorously with your forefinger several times, then tap the end of the tube on the table to make sure the contents are all at the bottom of the tube. Do NOT add plasmid to the Control tube. 10. Place both tubes on ice, and let them sit for 15 minutes. 11. Label the underside of your Luria broth (LB) ampicillin plates with your group s initials and the date. Label one 1 pgfp and the other Control. 271

4 Science & Global Issues/Biology genetics Sterile Technique Practices for Working with E. Coli 1. Keep all equipment away from your eyes and nose to avoid contact with bacteria. 2. Wipe all surfaces with a disinfectant solution before and after working with E. coli. 3. Wash your hands before and after any work with E. coli. 4. Treat all equipment that has been exposed to E. coli (pipettes, spreaders, microtubes, etc.) by soaking them in a 10% bleach solution for 10 minutes before placing them in the appropriate waste container. 5. To prevent overgrowth of E. coli do not over-incubate culture plates. 12. The next step is to shock the bacteria with heat. Heat shock causes the bacteria cells to take in the plasmid with the GFP gene. Rub your hands together rapidly to be sure they are warm. Remove both tubes from the ice, and hold them in your hands to incubate them. Shake your hands gently for 2 3 seconds to mix the fluid in the tubes. Incubate EXACTLY 3 minutes, and immediately put the tubes back on ice for 1 minute. 13. With a new sterile pipette transfer 100 μl of the mixture from the Control tube to the control plate. Use one of the spreaders to spread the liquid across the entire plate, taking care not to damage the agar. Discard the pipette in the waste container. 14. With another new sterile pipette and the other spreader, repeat Step 13 to transfer the contents of the 1 pgfp tube to the 1 pgfp plate. 15. Let the plates sit for 3 minutes to allow the agar to absorb the liquid that contains the bacteria. 16. Turn the plates upside down. Your teacher will give you instructions for storing the plates. 17. On Student Sheet 2.2, E. coli Growth Observations, record your observations of each plate on the section of the Student Sheet designated, time 5 0 hours. 18. After 48 to 72 hours, observe the plates under a UV light. Keep the lids on the plates. Do not look directly at the UV light source, as it might damage your eyes. In your science notebook, record your observations of the plates your team prepared. Be sure to record the number of bacterial colonies on each plate, and to sketch each plate and the colonies. 272

5 creating Genetically Modified bacteria Activity Following your teacher s instructions, compare the results from your plates with the number of colonies on plates that other groups grew. 20. Dispose of all materials as instructed by your teacher, and sterilize your table surface. 21. Wash your hands thoroughly with soap and water. 22. Throughout this unit, you will read case studies about genetic modification. You will use the information you collect on Student Sheet 2.3, Genetics Case Study Comparison, to answer Analysis Questions in a number of activities. 23. Follow your teacher s directions for reading the case study about biofuels. As you read, follow the Read, Think, and Take Note strategy. To do this: Stop at least three times during the reading to mark on a sticky note your thoughts or questions about the reading. Use the list of guidelines below to start your thinking. After writing a thought or question on a sticky note, place it next to the passage in the reading that prompted your note. Discuss with your partner the thoughts and questions you had while reading. 24. Complete the information for Biofuels from Bacteria on Student Sheet 2.3, Genetics Case Study Comparison. Read, Think, and Take Note: Guidelines As you read, from time to time, write one of the following on a sticky note: Explain a thought or reaction to something you read. Note something in the reading that is confusing or unfamiliar. List a word that you do not know. Describe a connection to something you learned or read previously. Make a statement about the reading. Pose a question about the reading. Draw a diagram or picture of an idea or connection. 273