1 | Page Bacterial Transformation Lab Learning Objectives: • Enable ...

is recovered. This method has enabled scientists to obtain large quantities of more than 1,000specific genes, including the genes for human interferon, insulin, and growth hormone.DNA technology has triggered advances in almost all fields of biology by enabling biologists totackle specific questions with finer tools. By far the most ambitious research project made possibleis the Human Genome Project, a multibillion-dollar effort to determine the nucleotide sequence ofthe entire human genome. The entire human genome will be characterized by cloning in specializedvectors and by analyzing the clones to determine where their inserted DNA molecules reside on the23 chromosomes of the human genome. Such an analysis, aided by research on smaller genomes,produces a physical map of all the genes in the human genome. This map will be pivotal in researchconcerning the diagnosis of human genetic disorders and other diseases, the application of genetherapy, and the development of vaccines and other pharmaceutical products.DNA technology also offers applications to the study of forensic science, problems relating to theenvironment, and the ever-growing need for better, more productive agriculture. DNA testing hasrefined the process of forensic analysis because not as much tissue is required and guiltyindividuals can be identified with a higher degree of certainty. With recent advances in restrictionfragment length polymorphism (RFLP) analysis, polymerase chain reaction (PCR), and variablenumber of tandem repeats (VNTRs), the probability that two people will have exactly the same DNAfingerprints is very small. (For legal cases the probability is between one chance in 100,000 and onein a billion, depending on the number of genomic sites compared.)Environmental research has seen an increase in the applications of genetics engineering.Microorganisms are being used to extract heavy metals from mining waste; clean up theenvironment; provide another source for metals such as lead, copper, and nickel; recycle wastes;and detoxify toxic chemicals. Sewage treatment facilities rely on the ability of microbes to degrademany organic compounds into nontoxic forms. Chlorinated hydrocarbons are a real problem, andbiotechnologists are attempting to engineer microorganisms that can be incorporated into thetreatment process to degrade these compounds. Eventually the microorganisms may beincorporated into the manufacturing of these toxic chemicals, thus preventing their release aswaste.Agricultural applications of DNA technology include research in the areas of animal husbandry andmanipulating plant genes. Products of recombinant DNA methods are already being used to treatfarm animals. These include new or redesigned vaccines, antibodies, and growth hormones. Bovinegrowth hormone (BGH), made by E. coli, increases milk production by approximately 10% in milkcows and improves weight gain in beef cattle. Transgenic organisms (organisms that contain genesfrom other species) have been developed for potential agricultural and medical uses (e.g., organtransplants, production of human hormones, etc.). Genetic engineers working with plant genescommonly use DNA vectors to move genes from one organism to another. Several companies havedeveloped strains of wheat, cotton, and soybeans carrying a bacterial gene that makes the plantsresistant to herbicides used by farmers to control weeds. Tomatoes containing genes that retardspoilage have been approved by the Food and Drug Administration (FDA). A number of crop plantshave been engineered to be resistant to infectious pathogens and pest insects. Increasing the abilityof bacteria to “fix” nitrogen or transferring the ability to fix nitrogen directly to plants would reduceor eliminate the need for expensive and environmentally detrimental fertilizers.Safety, Handling, and Disposal2 | P a g e

It is your responsibility to specifically follow your institution’s standard operating procedures(SOPs) and all local, state, and national guidelines on safe handling and storage of all chemicals andequipment you may use in this activity. This includes determining and using the appropriatepersonal protective equipment (e.g., goggles, gloves, lab coat). If you are at any time unsure aboutan SOP or other regulation, check with your instructor. Dispose of used reagents according to localordinances.When dealing with biological materials, take particular precautions as called for by the kitmanufacturer or supplier. A commensal organism of Homo sapiens, E. coli is a normal part of thebacterial fauna of the human gut. It is not considered pathogenic and is rarely associated with anyillness in healthy individuals. Adherence to simple guidelines for handling and disposal makes workwith E. coli a nonthreatening experience for instructor and students.Wear gloves when handling bacteria. When pipetting a suspension culture, keep your nose andmouth away from the tip end to avoid inhaling any aerosol that might be created.Avoid overincubating plates. Because a large number of cells are inoculated, E. coli is generallythe only organism that will appear on plates incubated for 12–24 hours at 37°C. However, withlonger incubation, contaminating bacteria and slower-growing fungi may arise. If students will notbe able to observe plates following initial incubation, refrigerate plates to retard growth ofcontaminants.Always reflame the inoculating loop or cell spreader one final time before setting it down on thelab bench. Wipe down lab benches with 10% bleach solution, soapy water, or disinfectant (such asLysol) at the end of the activity. Wash your hands before leaving the laboratory.General ProcedureIn this exercise, plasmid lux and a control plasmid (pUC18) will be introduced into E. coli bytransformation. There are four basic steps to the procedure:Treat bacterial cells with CaCl 2 solution in order to enhance the uptake of plasmid DNA. SuchCaCl 2-treated cells are said to be “competent.” (This step should be performed by the instructorbefore or during the laboratory session.) Incubate the competent cells with plasmid DNA. Selectthose cells that have taken up the plasmid DNA by growth on an ampicillin-containing medium.Examine the cultures in the dark.ProcedureA. Preparation of competent cells (These steps were performed by the instructor)1. Place a vial of CaCl 2 solution and the tube of E. coli in the ice bath.2. Using a sterile pipet, transfer 590 µL CaCl 2 solution to the tube containing 50 µL of the bacteria.3. Tap the tube with the tip of your index finger to mix the solution.4. Incubate the cells for at least 10 minutes on ice. The cells are then called competent becausethey can take up DNA from the medium. If desired, the cells can be stored in the CaCl 2 solutionfor 12–24 hours.B. Uptake of DNA by competent cellsNote: There are 2 plasmids involved in this experiment each group will only use 1 of the 2plasmids. The instructor will assign which plasmid your group will use.3 | P a g e

1. Label one small Eppendorf tube “C” (for control plasmid DNA) or one tube “lux” (for plasmid luxDNA)2. Place both tubes in an ice bath.3. Using a sterile micropipette, add 5 µL control plasmid to the tube labeled “C” or 5 µL plasmid luxto the tube labeled “lux”. Make sure to keep all tubes in the ice until instructed otherwise.4. Gently tap the tube of competent cells with the tip of your index finger to ensure that the cellsare in suspension.5. Using a sterile transfer pipet, add 70 µL of the competent cells to each of the two tubes.6. Tap each of these tubes with the tip of your index finger to mix these solutions, and store bothtubes on ice for 15 minutes. During this time, one member of the group should obtain oneadditional tube. Add 35 µL of competent cells to each tube and label the tubes “NP” (noplasmid). Every group will have a no plasmid tube assigned to them.7. HEAT SHOCK: Transfer all the tubes to a water bath preheated to 37ºC, and allow them to sit inthe bath for 5 minutes. Make sure you are able to identify which tubes below to your groupbefore you place them in the water bath.8. Use a sterile pipet to add 275 µL nutrient broth to the control and lux tubes and 150 µL ofnutrient broth into the no plasmid tube.9. Incubate the tubes at 37°C for 45 minutes. Use this time to make your prediction (Table 1) andanswer questions.C. Selection of cells that have taken up the plasmid by growth on an ampicillin containing mediumNote: There are a total of 6 plates per trial. Each group will be working with 3 plates. Theinstructor will assign the plates your group will work with.1. Each group will obtain 3 agar plates from the instructor. Label the plates as indicated in Figure1. Keep in mind the instructor assigned your group the treatments you will be working with.2. Using a sterile pipet, remove 130 µL mixed bacterial suspension from the “C” tube,remove the lid from the “Control” plate, and dispense the bacteria onto the agar. Use a cellspreader to spread the bacteria evenly onto the agar surface.3. Transfer 130 µL bacterial suspension from the “lux” tube to the “lux” plate and spreadthese cells onto the agar surface as described in the previous step.4. Cells from the tubes that did not contain plasmids (NP) should be plated onto two plates(NP) as described in steps 2–3.5. Replace the lids on the plates, and leave the plates at room temperature until the liquid has beenabsorbed (about 10 minutes).6. Invert the plates and incubate them at 37°C.D. Examine cultures in the dark1. Retrieve your group’s plates from the refrigerator.2. Open each plate, one by one, to determine if E. coli growth occurred. If growth occurred, notethe growth type (lawn or colonial). Record your results in Table 2.3. Allow at least 3 minutes for the eyes to adjust to the dark in a light-free room. View your platesand the plates of your classmates in the dark and then in the light. Record your results in thefollowing table. Were the results as expected? Explain possible reasons for variations fromexpected results (Table 2).4. Calculate Transformation Efficiency and answer questions.Data Analysis:4 | P a g e

PREDICTIONS:TreatmentExpected Result(Growth or No Growth)Bioluminescence(yes or no)Reason For ExpectationsLB cLB/Amp cLB -LB/Amp -LB/Amp luxLB luxLB c LB/Amp c LB -LB lux LB/Amp lux LB/Amp -5 | P a g e

‣ Based on your predictions, state your null and alternative hypotheses regarding what youexpect to see if the bacteria plated on the ampicillin rich medium were successfullytransformed.Questions:1. What role did the following compounds or steps play in bacterial transformation?a. Calcium Chlorideb. Heat Shockc. Agard. LB Brothe. Ampicillinf. Plasmidg. Aseptic Techniques2. What are you selecting for in this experiment? (i.e., what allows you to identify whichbacteria have taken up the plasmid?)3. What does the phenotype of the transformed colonies tell you?4. What one plate would you first inspect to conclude that the transformation occurredsuccessfully? Why?6 | P a g e

5. In nature, DNA uptake by different organisms can impart advantages as well asdisadvantages to the host organism.a. When would genetic transformation be advantageous to a host organism?b. When would genetic transformation be maladaptive to a host organism? Whatconsequences would there be for the host cells?Results:c. Can you think of a case where the uptake of foreign DNA would be advantageous forthe organism, but disruptive for the surrounding ecosystem?TreatmentObserved Growth TypeBioluminescence(yes or no)Reasoning for observedresultsLB cLB/Amp cLB -LB/Amp -LB/Amp luxLB lux7 | P a g e

LB c LB/Amp c LB -LB lux LB/Amp lux LB/Amp -Transformation Efficiency6. The total amount (µg) of plasmid DNA used can be calculated with the following formula.µg DNA = concentration (µg/ µL) of DNA x volume of DNA (µL)7. Calculate the total volume of cell suspension prepared in the control DNA tube.Total volume (µL) = amount (µL) of plasmid + amount (µL) of LB8 | P a g e

8. Since only a portion of the control DNA solution was added to the LB/Amp C plate, you willneed to calculate the fraction of DNA spread onto this plate using the formula below:( ) ⁄( )9. Using the values obtained for questions 3-5, determine the actual amount of DNA (µg)present on your plateTotal amount (µg) of DNA = µg of DNA x Fraction of DNA spread10. Calculate transformation efficiency⁄⁄11. Repeat questions 3-7 to calculate the transformation efficiency for the ⁄9 | P a g e

12. Compare and contrast the number of colonies on each of the following pairs of plates. Whatdoes each pair of results tell you about the experiment?a. LB C and LB -b. LB/Amp – and LB Cc. LB/Amp C and LB/Amp -d. LB/Amp C and LB Ce. LB/Amp lux and LB –f. LB/Amp lux and LB/Amp -g. LB/Amp C and LB/Amp luxh. LB/Amp lux and LB C13. Do your results agree with your predictions in Table 1? Explain.14. What factors could have affected transformation efficiency?10 | P a g e