Transformation Lab

Transformation Lab Genetic transformation occurs when a cell takes up (i.e. takes inside) and expresses a new piece of genetic material—DNA. Genetic transformation literally means change caused by genes and it involves the insertion of a gene(s) into an organism in order to change the organism’s traits. Remember that a gene is a piece of DNA which provides the instructions for making (coding for) a particular protein. 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 Rfp amp gene therapy; that is, by genetically transforming a sick person’s cells with healthy copies of the gene involved in their disease. Another medical application is in the creation of proteins, such as insulin (synthesized by Genentech) and factor VIII (blood clotting protein synthesized by Bayer). 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 just as they would make their own proteins. This insulin can then be used to treat patients with the genetic disease, Diabetes, whose insulin genes do not function properly. In this lab, you will learn about the process of moving genes form one organism to the other with the aid of a plasmid. In addition to one large circular chromosome which contains all of the genes a bacterium needs for its normal existence, bacteria naturally contain one or more tiny circular pieces of DNA called plasmids. Plasmid DNA contains genes for traits that may be beneficial to bacterial survival under certain environmental conditions. In nature, bacteria can transfer plasmids back and forth, allowing them to share these beneficial genes. This mechanism allows bacteria to adapt to new environments. The recent occurrence of bacterial resistance to antibiotics is due to transmission of plasmids. In order to do transformation, the gene to be transferred is placed into a plasmid. This is done with the help of restriction enzymes, naturally occurring enzymes from bacteria that recognize a particular sequence of DNA bases and cut at that sequence. Bacteria use restriction enzymes to protect themselves from viruses which inject their DNA into the bacteria; the enzymes can cut the viral DNA before it can hurt the bacteria. The same restriction enzyme is used to cut the ends of the gene we want to transfer and to cut open the plasmid. Because the cuts are made at the same base sequence, the ends will match and reattach when placed together. The plasmid used in this lab is called pARA-R developed by Amgen Laboratories for use in the classroom. The plasmid is used as the vector (transport mechanism) that will transform a nonpathogenic (non-disease causing) E. coli bacteria. The pARA-R plasmid contains a gene for the Red Fluorescent Protein (RFP) that comes from the bioluminescent from a sea anemone, a soft-bodied animal related to coral and jellyfish. This gene allows the sea anemone to produce a protein that fluoresces, or glows, under ultraviolet light. The gene can be turned on in transformed cells by simply adding the sugar, arabinose, to the cells’ nutrient medium. Without arabinose, the bacteria will not fluoresce. So arabinose is the “switch” for the RFP gene in this plasmid. To move the plasmid through the E. coli cell membrane you will use a transformation solution of calcium chloride (CaCl2), and a procedure know as “heat shock” which will open small pores in the bacteria. In order to select only the bacteria that have received the new gene, the plasmid also has a gene for resistance to the antibiotic Ampicillin. The gene codes for the production of a protein, betalactamase, that allows the bacteria to digest the antibiotic before it can cause any harm. Therefore, if ampicillin is mixed into the agar on the bacterial plates, the only bacteria that can survive there will be bacteria with the gene/plasmid for ampicillin resistance. Since we know that the gene for Red Fluorescent Protein is on the same plasmid, if any bacteria grow on the plate, they must have been transformed, even if we can’t see them glow. Transformed cells will appear white and grow in circular colonies. If arabinose is present, these colonies will appear bright pink! Below is a map of the plasmid used in this lab. By examining the growth of bacteria under these conditions, you can verify that your procedure worked, and you can identify the bacteria transformed with the plasmid. How will you know if you are successful? The bacteria will have a new and highly visible trait: It will now produce red fluorescent protein, which makes the cells red or bright pink! With the addition of the Restriction enzymes and Ligase steps we will add the rfp gene to make Hind III BamHI Plasmid to be inserted BamHI BamHI Hind III Hind III TRANSFORMATION QUESTIONS: 1. If there are any genetically transformed bacterial cells, on which plate(s) would they most likely be located? Explain your predictions. 2. Why do scientists prefer to do transformation on a simple organism like bacteria? 3. What does transformation mean? 4. How is transformation used in agriculture? 5. How is transformation used in medicine? 6. Why do plasmids make good vectors (something that transfers DNA from one place to another)? 7. Describe how a strain of bacteria could develop antibiotic resistance. a. Why is this a problem? 8. What genes does the pARA-R plasmid carry? 9. What other molecule is required for expression of the RFP gene? BACTERIAL GROWTH PREDICTIONS 10. Predict how much bacterial growth you will see on each plate. Mark the plate/plate section with +++ (for high growth), ++ (for medium growth), + (low growth), or - (for no growth): Plate Contains: Table 1: P- Control Group (Non- Transformed Bacteria) Predicted Growth Conclusion if Predicted Conclusion if Predicted Growth occurs Growth Does Not Occur Luria Broth (LB) Luria Broth Ampicillin (LB/amp) Table 2: P+ Experimental Group (Transformed Bacteria) Plate Contains: Luria Broth (LB) Luria Broth Ampicillin (LB/amp) Luria Broth ampicillin arabinose (LB/amp/ara) Predicted Growth Conclusion if Predicted Growth occurs Conclusion if Predicted Growth Does Not Occur Student Transformation Lab Protocol Period_____ Individuals in my lab group and their assigned tasks: NAME TASK (me) COMPLETED Materials The following will be given by teacher: Microtube of plasmid with rfp gene (RP) Microtube of Luria Broth (LB) Microtube of competent E. coli cells (CC) o Note: The CC tube must be kept on ice at all times. P-20 micropipette P-200 micropipette Tip box of disposable pipette tips 42°C water bath with floating microfuge tube rack (will be shared among all groups) 37°C incubator (will be shared among all groups) Waste container with 10% bleach for materials that come into contact with E. coli cells (will be shared among groups) 3 Petri plates with agar: o 1 of LB o 1 of LB/amp o 1 of LB/amp/ara o 1 LB plate 2 1.5-mL microfuge tubes Permanent marker Styrofoam cup of Crushed ice Pack of cell spreaders (will be shared among groups) Timer (cell phone works) Colored tape (will be shared among groups) Plastic Pipet (steril) Checklist of materials at each lab station: Note: Fill a cup with some of the crushed ice with a little water. You’ll need to keep the CC tube on ice at all times. Checklist for Procedure: 1. Check to be sure you have all the needed materials at your lab station. 2. Label two clean microfuge tubes “P–” and “P+” along with your initials and class period. 3. Place the P– and P+ and tubes in the Styrofoam cup of ice. LAB TECHNIQUE: Bacterial transformation requires sterile techniques. It is essential that these directions be followed precisely. Steps 4 & 5, will be with the help of your teacher: 4. With the help of your teacher, using the large P-200 micropipette, add 50 μL competent cells from the CC tube to the P– and P+ tubes: a. Set the P-200 micropipette to 50 μL. b. Very carefully, re-suspend the bacterial cells in the CC tube by gently pumping the pipette two times in the solution. c. Add 50 μL of CC to each of the empty chilled tubes (P– and P+), holding each tube at its rim to keep it cold, and return each tube quickly to the ice. LAB TECHNIQUE: To avoid contamination, be sure to use a new micropipette tip for each addition. 5. Using a clean , add RP to the tube labeled “P+”: a. Set the P-20 micropipette to 10.0 μL. b. Hold the chilled P+ tube by the upper rim and add 10.0 uL of RP. Mix the solutions by pumping the pipette two times in the liquids, and return the P+ tube to the ice. 6. Keep the P– and P+ tubes on ice for 15 minutes. NOTE: During the 15-minute interval, do the following steps. 7. While the cells are on ice, prepare your three agar Petri plates—one plate each of LB has one stripe, LB/amp has two stripes, and LB/amp/ara has three stripes: a. Label the bottom of each plate (the part that contains the agar) with your group number and class period. Write small and on the edge of the plate. b. With the plates closed, draw a line on the LB plate and the LB/amp plate that divides each plate in the middle. Label half of each plate “P–” and the other half “P+.” Label the LB/amp/ara plate “P+.” The plates will be arranged as follows: 8. Following the 15 minute interval. Heat shock: Take your ice water bath with its tubes to the 42oC hot water bath. Transfer the tubes to the hot water and time for exactly 45 seconds. Make sure that the tubes are in contact with the hot water. Immediately return the tube to the ice and leave them there for at least a minute. 9. Using a transfer pipet, add 250 μL LB to the P– and P+ tubes. The line below the .5ml mark 10. If time permits, allow the cells in the P– and P+ tubes to incubate at room temperature for 15 minutes. 11. Open the P- tube, and with a clean pipette, gently pump the pipette two or three times in the P– tube to suspend the cells. Transfer about half of the cell mixture to the P- side of LB petri dish you prepared previously. Add the rest of this tube to the P- side of LB/amp petri dish. Do not discard the pipette. 12. With the same pipette, gently pump the pipette two or three times in the P+ tube to suspend the cells. Transfer about half of the mixture from the P+ tube to the LB/amp/ara P+ plate. 13. With the remainder of the mixture half will go to the LB P+ plate, and the rest to the LB/amp P+ plate. Bleach the pipette. 14. Spread the cells on each plate with a clean, sterile loop, to spread the liquid. Let this sit for a few minutes to absorb the liquid. LAB TECHNIQUE: Hold the spreader by the handle and do not allow the bent end to touch any surface, as this will contaminate the spreader. Place the used spreader in the bleach. 15. Allow all three plates to sit right side up for five minutes. 16. Using provided tape, tape all three plates together and label tape with your group number and class period. 17. Place the plates in the 37°C incubator upside down to prevent condensation from dripping onto the gels. 18. Place all microtubes, pipette tips, and cell spreaders in the bleach. 19. Incubate the plates for 24–36 hours at 37°C. 20. Examine the plates and in your notebook record the amount of growth on each half. 21. Discard the Petri plates in the bleach when directed to do so. Transformation QUESTIONS 1. Look at the results of your transformation. Do your actual results match your predicted results? If not, what differences do you see, and what are some explanations for these differences? 2. How many red colonies were present on your LB/amp/ara plate? 3. Why did the red colonies only appear on the LB/amp/ara plate and not the LB/amp plate? 4. Recombinant plasmids are engineered so that they can replicate in the cell independently of the chromosome replication. Why is it important to have multiple copies of a recombinant plasmid within a cell? 5. How is the information encoded in the rfp gene expressed as a trait? The rfp gene on DNA is 702 nitrogen bases long, use what you have previously learned about gene expression and the relationship between DNA, RNA, proteins to show how the start of this protein was made. DNA: mRNA: AA: TAC AAG CTT GCA TGC CTG CAG