General Biology I The Unity of Life Laboratory Genetic Transformation of Bacteria with pglo 10% of lab mark (2% of final course mark) modified from: BioRad Biotechnology Explorer pglo Bacterial Transformation Kit. This lab activity is intended to provide an opportunity for students to: become acquainted with some contemporary biotechnology procedures; apply links between technology and the theories of genetics; gain experience analyzing data and drawing simple conclusions from those data. Students will work in teams of three. Objectives: This lab aims to contribute to the development of the following components of the Cégep Champlain St. Lawrence Science Program Graduate Profile: I. Apply The Experimental Method II. Take A Systematic Approach To Problem Solving III. Use The Appropriate Information Technologies IV. Reason Logically V. Communicate Effectively VI. Learn In An Autonomous Manner VII. Work As Members Of A Team VIII. Make Connections Between Science, Technology and Social Progress X. Become Familiar With the Context in Which Scientific Concepts are Discovered and Developed XI. Develop Attitudes Appropriate For Scientific Work XII. Apply What They Have Learned To New Situations This lab also contributes to the attainment of the following elements of the 00UK objective: 1. To recognize the relationships between the structures and functions of certain levels of organization of living beings. 2. To analyze the mechanisms that are responsible for the genetic variation of living beings. This lab also contributes to the development of the following performance criteria of the 00UK objective: Proper use of concepts and terminology Clear description of the principal steps of a biological process. Accurate description of structures and their functions. Description of the correlations between structures and functions. Appropriate use of the laws of genetics and the chromosome theory of heredity. Observance of the experimental method and, where applicable, the experimental procedure. Adherence to safety and environmental protection regulations. Appropriate use of techniques of observation and experimentation. Pre-lab Assignment Due October 7, 2008 Each student is required to answer the following questions concerning the lab protocol: 1. On which plate(s) do you expect non-transformed bacteria will form colonies? Justify your prediction. (2 points) 2. On which plate(s) do you expect genetically-transformed bacteria will form colonies? Justify your prediction. (2 points) 3. Identify the positive and negative control plate(s). Explain the function of each. 4. The pglo plasmid contains many genes. What is the role of the following genes? a. ori gene b. GFP gene c. bla gene d. arabinose promoter. 1
Background Information: Genetic transformation occurs when a cell takes up (takes inside) and expresses a new piece of genetic material DNA. This new genetic information often provides the organism with a new trait which is identifiable after transformation. Genetic transformation literally means change caused by genes and involves the insertion of one or more gene(s) into an organism in order to change the organism s traits. Genetic transformation is used in many areas of biotechnology. In agriculture, genes coding for traits such as frost, pest, or drought resistance can be genetically transformed into plants. In bioremediation, bacteria can be genetically transformed with genes enabling them to digest oil spills. In medicine, diseases caused by defective genes are beginning to be treated by gene therapy; that is, by genetically transforming a sick person s cells with healthy copies of the defective gene that causes their disease. Genes can be cut out of human, animal, or plant DNA and placed inside bacteria. For example, a healthy human gene for the hormone insulin can be put into bacteria. Under the right conditions, these bacteria can make authentic human insulin. This insulin can then be used to treat patients with the genetic disease, diabetes, because their insulin genes do not function normally. The pglo System You will use a simple procedure to transform bacteria with a gene that codes for Green Fluorescent Protein (GFP). The real-life source of this gene is the bioluminescent jellyfish Aequorea victoria, and GFP causes the jellyfish to fluoresce and glow in the dark. Following the transformation procedure, the bacteria express their newly acquired jellyfish gene and produce the fluorescent protein which causes them to glow a brilliant green color under ultraviolet light. In this activity, you will learn about the process of moving genes from one organism to another with the aid of a plasmid. In addition to one large chromosome, bacteria naturally contain one or more small circular pieces of DNA called plasmids. Plasmid DNA usually contains genes for one or more traits that may be beneficial to bacterial survival. In nature, bacteria can transfer plasmids back and forth, allowing them to share these beneficial genes. This natural mechanism allows bacteria to adapt to new environments. The recent occurrence of bacterial resistance to antibiotics is due to the transmission of plasmids. The pglo plasmid contains the gene for GFP and a gene for resistance to the antibiotic ampicillin. pglo also incorporates a special gene regulation system that can be used to control expression of the fluorescent protein in transformed cells. The gene for GFP can be switched on in transformed cells simply by adding the sugar arabinose to the cell s nutrient medium. Selection for cells that have been transformed with pglo DNA is accomplished by growth on antibiotic plates. Transformed cells will appear white (wild-type phenotype) on plates not containing arabinose, and fluorescent green when arabinose is included in the nutrient agar. You will be provided with the tools and a protocol for performing genetic transformation. Your task will be: 1. To do the genetic transformation. 2. To determine the degree of success in your efforts to genetically alter an organism. 2
Microbial Transformation with pglo October 7 and 21 Required Materials E. coli starter plate 4 agar plates (1 LB, 2 LB/amp, 1 LB/amp/ara) transformation solution (CaCl2) LB nutrient broth inoculation loop sterile, transfer pipets microtube holder container full of crushed ice 2 microtubes Rehydrated pglo plasmid 42 C water bath 37 C incubator P-200 micropipet and tips biological waste bags glass hockey stick EtOH in watch glass Procedure: Transformation October 7 1. Use a waterproof marker to label 2 microtubes with your team s name and the lab time. Mark one microtube + and the other microtube with a. Place labeled tubes in the microtube rack. 2. Open the tubes and, using a sterile transfer pipet, transfer 250 μl of transformation solution (CaCl2) into each microtube. Place on ice. Discard pipet. 3. Use a sterile inoculation loop to pick up a SINGLE colony of bacteria from your starter plate. Immerse the bacteria into the liquid within the tube marked +. 4. Spin the loop between your index finger and thumb until the entire colony is dispersed in the transformation solution (with no floating chunks). Replace the tube in the rack in the ice. Sterilize the loop before you set it back down. 5. Examine the bacterial colonies on the E. coli starter plate. Record size and color of the colonies in the Microbial Transformation Data Sheet. 6. Using a sterile inoculation loop, add a single bacterial colony to the solution in the tube marked. Spin to disperse the colony as in step 4. Sterilize the loop. 7. Examine the solution within the tube marked + with the UV lamp. Note your observations. Note: 8. Immerse a sterilized inoculation loop into the pglo plasmid DNA stock tube. Withdraw a loopful. There should be a film of plasmid solution across the ring. This is similar to seeing a soapy film across a ring for blowing soap bubbles. Mix the loopful into the cell suspension of the + tube. Close the tube and return it to the rack on ice. Sterilize the loop before you set it back down. 9. Close the tube marked. Add nothing to this tube. 10. Incubate both microtubes on ice for 10 minutes. Make sure the tubes are in contact with the ice. 11. Label four LB agar plates on the bottom (not the lid) with the date, your team s initials, the lab time and: Label one LB/amp plate: + DNA Label the LB/amp/ara plate: + DNA Label the other LB/amp plate: DNA Label the LB plate: DNA 3
12. After the 10 minute incubation on ice has ended, heat shock the cells. Remove the rack containing the microtubes from the ice and place it in a 42 C water bath for EXACTLY 50 seconds. Make sure the tubes are in contact with the water. Note: For the best transformation results, the transfer from the ice (0 C) to 42 C and then back to the ice must be rapid. It is best to bring the ice bucket over to the water bath to minimize the amount of time the solutions are exposed to room temperature. 13. Incubate tubes on ice for 2 minutes. 14. Remove the rack containing the microtubes from the ice and place on the bench top. 15. Use a sterile transfer pipet to add 250 μl of LB nutrient broth to the tube marked +. Discard the pipet in the biological waste bag. 16. Use a new sterile transfer pipet to add 250 ul of LB to the other tube. Discard the pipet in the biological waste bag. 17. Close the lids of both tubes and incubate the tubes for 10 minutes at room temperature. 18. Tap the closed tubes with your finger to mix. 19. Use a P-200 micropipet (with sterile tip) to aseptically transfer 100 µl of + solution to each of the 2 agar plates labeled +DNA. Discard the contaminated pipette tip into a biological waste bag. 20. Use a P-200 micropipet (with sterile tip) to aseptically transfer 100 µl of solution to each of the 2 agar plates labeled DNA. Discard the contaminated pipette tip into a biological waste bag. 21. Use a sterilized hockey stick to evenly spread the liquid bacterial culture over the surface of the agar. Sterilize the hockey stick between agar plates. Dip a bent glass rod into ethanol in a watch glass and pass the rod through the flame. The ethanol will quickly burn off and the glass rod will be sterilized. After the sterilized glass rod has cooled, use it to evenly spread the liquid bacterial culture over the surface of the agar. Re-sterilize and cool the glass rod before putting it down. 22. Stack up your plates and tape them together. Place the stack of plates upside down in the 37 C incubator for 24 hours. Procedure: Data Collection October 21 1. Observe the results you obtained from the transformation lab under normal room lighting. Record your observations on the Microbial Transformation Data Sheet. 2. Turn out the lights and hold the UV lamp over the plates. Carefully observe and draw what you see on each of the four plates on the Microbial Transformation Data Sheet 3. For each plate, record, relative bacterial growth number of bacterial colonies color of bacterial colonies colony size 4. Determine the number of green fluorescent colonies growing on the LB/amp/ara plate. Record this number in the class data sheet. 4
Procedure: Calculate transformation efficiency Determine the transformation efficiency for your group and the average efficiency for all groups. group transformation efficiency = total number of cells growing on the LB/amp/ara plate amount of DNA (0.16 μg) spread on the agar plate average transformation efficiency = average number of cells growing on the LB/amp/ara plate amount of DNA (0.16 μg) spread on the agar plate Assignment one per team Due: November 4 1. Submit a completed Microbial Transformation Data Sheet. 2. Calculate the transformation efficiency for your data as well as the overall transformation efficiency using the class data. Answer the following questions: 3. Which traits seem to be the same in the transformed E. coli and the non-transformed E. coli? 4. Which traits of the bacteria have changed in the transformed bacteria? Compare the traits of E. coli that were not transformed with the E. coli that were transformed. 5. Based on the results obtained, how could you prove that the changes that occurred were due to the procedure that you performed? 6. Very often an organism s traits are caused by the interaction of its genes and its environment. For the pglo experiment, what two factors must be present in the bacteria s environment for you to observe the green color trait? This is an example of what type of gene regulation? Explain. 7. What advantage would there be for an organism to able to turn on or off particular genes in response to certain conditions? 8. Why is it necessary to incubate the transformed cells for 10 minutes at room temperature before spreading them on the plates containing antibiotic? Consider how the bacterial cells in the pglo procedure are able to resist the ampicillin as well as the steps of gene expression. 5