Antibiotic-Resistant Bacteria

Transcription

1 Antibiotic-Resistant Bacteria You may be familiar with the antibiotics that are prescribed to you by your doctor to help you overcome a bacterial infection. While some antibiotics are synthesized in the lab by drug companies, the first therapeutic antibiotics were produced by bacteria. These first therapeutic antibiotics were substances that some bacteria naturally used to protect themselves from other bacteria, and there is ongoing research to find new bacterially-generated antibiotics. You may be wondering, how do antibiotics work? Natural antibiotics kill competing bacteria by interfering with vital cellular processes, such as cell wall synthesis or protein synthesis, that are essential for survival and reproduction of those bacteria. Scientists and physicians have discovered that those same antibiotics can be used to fight a variety of bacterial infections. There is a weakness in antibiotic therapy; some bacteria are resistant to certain antibiotics, so these antibiotics do not have an effect on them. Often, antibiotic resistance is exhibited by bacteria in response to their own antibiotic compound. However, antibiotic-resistance can also be an acquired trait, meaning the bacteria have evolved (altered their genomes) to survive in the presence of antibiotics produced by competing bacteria. For example, if bacteria-x has developed resistance to antibiotic-y but not to antibiotic-z, then bacteria-x will survive in the presence of antibiotic-y and die in the presence of antibiotic-z. In this lab, we will test the acquired antibiotic resistance of multiple types of bacteria (types are referred to as strains ). You will be provided with a culture of Escherichia coli (E. coli) containing genes that confer resistance to only one antibiotic. The antibiotics that we will use in this experiment are ampicillin, kanamycin, and chloramphenicol. We will use a variety of laboratory techniques to help us determine which of the above antibiotics our culture of E. coli is resistant to therefore, we will determine its antibiotic resistance. The techniques we will use are (1) a sequence of DNA extraction, PCR, and gel electrophoresis; and (2) a qualitative analysis of antibiotic resistance, using bacterial Page 1

2 cultures incubated with antibiotics. If the protocol is carefully followed, both techniques should show similar results. IMPORTANT SAFETY INFORMATION BEFORE YOU BEGIN You will work with microorganisms in this laboratory sequence. This work should not be harmful to you, but you must learn to use basic aseptic technique and follow laboratory safety protocols. Please remember these guidelines: 1) You MUST handle your own specimens at all times! Please do not share specimens across groups without approval from your instructor. You should wear gloves every time you work on this project. 2) Before and after every laboratory session, you should wipe down your benchtop with 70% ethanol and allow it to air dry this will remove potential contaminating bacteria from your workspace. If one is available, please wear a laboratory coat for each session. 3) At the end of each day, you should thoroughly wash your hands before leaving the lab area. 4) Remember: If at any time you have questions, ask before you dispose of any specimens or supplies. Within UF laboratories, used gloves should always be discarded in red biohazard bags. Page 2

3 Background Polymerase chain reaction (PCR) is a technique that allows scientists to amplify sequences of DNA, so that the DNA can be visually analyzed in a gel cassette (after running gel electrophoresis). We will be performing PCR on the E. coli genome of your selected culture to amplify the genes that confer antibiotic resistance. Gene amplification works using the given genome you have attained: first, the DNA is denatured (split apart into two complementary strands); then, the single-stranded DNA is used as a template for in vitro synthesis of new DNA (by a polymerase enzyme) to make millions of copies of that same gene. By making many copies of the gene, we can visualize the DNA after performing gel electrophoresis. Gel electrophoresis is the process of separating macromolecules based on their electrical charges, using an electric current. To summarize, the copied DNA is placed in wells (indentations) on one side of an agarose gel, and an electric field is applied to the plate where the gel is located. The electric field pulls the negatively-charged DNA strands through the gel towards the opposite side; because of the different lengths (sizes) of genes, larger DNA strands (larger genes) move slower than smaller DNA strands (smaller genes) though the agarose gel. This allows us to semiquantitatively compare the amount of a gene present across a group of bacterial genomes. Page 3

4 Materials: For each portion of the experiment, you will need the following: Isolation of E. coli DNA o Gloves o Ice bucket (for storage until PCR) o Sharpies (Extra Fine or Ultra Fine tips, for writing on caps) o Sterile plastic squeeze pipette (2 per group of 2) o Two each, 2-mL microcentrifuge tubes o Microcentrifuge tube rack (shared per group of 4 students) o Pipettes (P1000, P200, P20, P10) and pipette tips o Chelator beads solution (Instagene matrix) (200 ul per tube) o Vortex o Heat block with water in each well (turn on and heat to 100 C, 30 minutes early) PCR: o E. coli: 2 tubes containing isolated bacterial DNA o Gloves o Ultra Fine point sharpie o Ice bucket o Thermocycler o 1 sterile microcentrifuge tube o 7 each PCR tubes per group o Sterile water (one bottle per group of 2 students) o Pipettes (P200, P20) and pipet-tips (P200, P20) o PCR tube rack o GoTaq Hot Start Green Master Mix o Forward and Reverse Primers (three sets; one for each antibiotic-resistance gene) o Microcentrifuge tube rack Gel Electrophoresis: o Your PCR reactions from last time o Pipette tips for P20 pipette o Pipette tips for P200 pipette o Pipette tips for P1000 pipette o P20 pipette o P200 pipette o P1000 pipette o 1.2% Agarose E-gel o 1kb ladder o Nuclease-free or sterile water Page 4

5 Protocol Isolation of E. Coli DNA 1. Spray 70% ethanol on the benchtop surface. Put on your gloves. Acquire your E. coli culture in broth (prepared for you by your instructor), along with the other materials you need. Tighten the lid on the tube of broth culture, then vortex to suspend the bacteria that might have settled to the bottom of tube. 2. Using a sterile (plastic) squeeze pipette, add 2 ml of broth (1 ml, at a time) from your culture to a 2- ml microcentrifuge tube. Label the tube. Close the cap securely and set this tube aside in a tube rack. 3. Place your tube in the microcentrifuge at your bench, making sure that your tube is balanced with other tubes located in opposite positions within the rotor. Centrifuge at max speed for 2 minutes to pellet the cells to the bottom of the tubes. Wait 30 seconds before opening the microcentrifuge to allow any microaerosols to settle before you open the lid. Hold your tube up to the light to make sure you can see a pellet of cells on the bottom of each tube (should look like a clump or smear). 4. Gently pipette off the liquid (NOT the cells on the bottom of the tube) from the microcentrifuge tube into your biohazard bag by using a p1000 pipette. Do not shake the tube. Be careful not to disturb the cell pellet at the bottom. A small amount of liquid will remain in the tube. If you accidentally pipette up the pellet of cells, dispense what is in the pipette back into the microcentrifuge tube and then centrifuge again to reform the pellet. 5. You will use the p1000 pipette (set at 200 ul) and an appropriate tip to transfer 200 ul of chelator beads solution to the cell pellet. Chelator beads will grab up any metal ions and cellular debris in a solution that might interfere with the PCR reaction you will be performing in the next lab period. To use the chelator beads, shake the beads solution to re-suspend the beads they settle to the Page 5

6 bottom very quickly! Before the beads settle to the bottom again, transfer 200 ul to the tube containing your cell pellet. Dispose of the pipette tip after a single use into the biohazard bag. 6. Mix the cells with the chelator beads by vortexing at high speed for 60 seconds, or by flicking the end of the tube roughly with your finger (your instructor will show you how to do this). 7. Place the tube in a 100 C heat block for 10 minutes. This will burst open the cells, releasing the DNA. 8. As in step 6, mix the cells again with the chelator beads by vortexing at high speed for 60 seconds, or by flicking the end of the tube roughly with your finger. 9. Place the tube in a balanced configuration in the microcentrifuge rotor, and centrifuge at max speed for 2 minutes to pellet the chelator beads at the bottom of the tube. Wait 30 seconds before opening the microcentrifuge lid to allow any microaerosols to settle. After centrifuging, the supernatant will contain your DNA. DNA is very light, so the cell debris and chelator beads will fall to the bottom and the DNA will remain suspended in the liquid. 10. Obtain an additional sterile microcentrifuge tube and label it as you did for the first tube. Place it into the tube rack. You will transfer the DNA from step 9 into the new tube for storage until next time. Use the p200 pipette (set at 100) and a sterile tip to transfer 100 ul of your DNA from step 9 into your new sterile microcentrifuge tube. Be careful not to transfer the chelator beads or any of the cell debris from the bottom of the tube. Dispose of the used tip in the biohazard bag. 11. Place the labeled tube with your transferred sample into the rack indicated by your instructor for storage at -20 C until next time. Alternatively, continue directly to the next section. 12. Dispose of your used microcentrifuge tubes into the biohazard bags as directed by your instructor, along with your used gloves. Spray down your benchtop with 70% ethanol solution. Be sure to wash your hands before you leave the lab area today. Page 6

7 DNA Polymerase Chain Reaction 1. Spray your benchtop with 70% ethanol before beginning your work. Put on your gloves; these are worn today to keep you from getting into your PCR tubes. Each group should label three respective PCR tubes as primer mix A, primer mix K and primer mix C. Add 30µl of H 2 O, 15µl of Forward Primer A (FP) and 15µl of Reverse Primer A (RP) to primer mix A; Add 30µl of H 2 O, 15µl of Forward Primer K (FP) and 15µl of Reverse Primer K (RP) to primer mix K; Add 30µl of H 2 O, 15µl of Forward Primer C (FP) and 15µl of Reverse Primer C (RP) to primer mix C. Your primer mix tubes are now complete and will provide sufficient volume for all reactions for your group. Place the tube into the ice bucket. Primer Mix (PCR tube) 1) 30 ul H2O 2) 15 ul Forward Primer (FP) 3) 15 ul Reverse Primer (RP) 2. For the other 5 PCR tubes, label the first four as E. coli A, E. coli K, E. coli C, and E. coli --. Label the last tube as control. To each of the corresponding three PCR tubes add 20µL of the primer mix made in the previous step and 5 ul of your sample DNA from the previous section. To the tube labeled E. coli --, add only 5µL of your sample DNA prepared previously (NO PRIMER MIX). Set aside in a PCR rack in an ice bucket. To the control tube, add 5 µl of sterile water. Page 7

8 DNA Sample A (PCR tube) 1) 20 ul of primer mix A 2) 5 ul of prepared DNA DNA Sample K (PCR tube) 1) 20 ul of primer mix K 2) 5 ul of prepared DNA DNA Sample C (PCR tube) 1) 20 ul of primer mix C 2) 5 ul of prepared DNA DNA Sample -- (PCR tube) 1) NO PRIMER MIX 2) 5 ul of prepared DNA 3. There is one more step to prepare your samples for PCR. ONLY complete this step immediately before the PCR reaction. You need to add MasterMix to all 5 of your group s PCR tubes. MasterMix contains a heat-stable DNA polymerase along with free nucleotides to allow PCR to occur; however, this mix cannot be held at room temperature for extended periods of time. There are only a few tubes of MasterMix in the lab they are found by the thermocyclers. Add 25 µl of MasterMix to each PCR tube (use a new tip each time!), making sure to pipette up and down to mix (try to avoid bubbles by pipetting slowly you must pipette to mix, do not stir). Deposit all of your group s PCR tubes in the thermocyclers and be sure to note which wells you placed them in. DNA Sample A Add 25 ul of Master Mix DNA Sample K Add 25 ul of Master Mix DNA Sample C Add 25 ul of Master Mix DNA Sample -- Add 25 ul of Master Mix IMPORTANT: Be sure to record in your notebook and MOST IMPORTANTLY on the 96-well grid sheet provided the location of your PCR tubes so that you can retrieve your samples next session. 4. Your samples will be run using the program listed below. Thermocycler program name: Cat2 Page 8

9 1. Primary melting and DNA polymerase activation: 94 C for 2:00 2. Denaturing step: 94 C for 45 seconds 3. Annealing step: 56 C for 40 seconds 4. Extension step: 73 C for 80 seconds 5. Repeat steps times 6. Final extension: 72 C for 2 minutes 7. Hold: 4 C indefinitely The expected product should be in the range of 500 bp 5. When finished setting up your PCR tubes, dispose of your gloves in the biohazard bag. Place your DNA tube back into the tube rack for your instructor to store. Please record here or in your laboratory notebook: Label of sample Location of sample in 96-well grid Page 9

10 Gel Electrophoresis (2 groups of 4 students will share a gel) 1. Spray your benchtop with 70% ethanol before beginning. Prepare the gel: Remove gel cassette from package and insert the gel (with comb in place) into the base, right edge first. The Invitrogen logo should be located at the bottom of the base. Press firmly at the top and bottom to seat the gel cassette in the PowerBase. A steady, red light will illuminate if the gel cassette is correctly inserted. Remove the comb from the gel. Then, obtain the materials that you will need for this experiment. 2. Gently mix your PCR reactions by flicking the tubes. Carefully pipette up 15µL of each sample and load it into the agarose gel as shown below. Carefully note which gel and which lane you have loaded your sample into. Either Group 1 or 2 can prepare the DNA Ladder lane. To prepare the DNA Ladder, add 8µl of the provided DNA Ladder Standard and 7µl H2O to a 1.5ml tube. Add all 15µl of this mixture to the DNA Ladder lane. Lane 2 15 ul negative control 1 Lane 3 15 ul negative control 2 If there are any remaining lanes in the gel, fill each of them with 15 ul of sterile water. 3. Press and release the 30 minute button on the E Gel PowerBase to begin electrophoresis. At the end of the run, the current will automatically shut off and the power base will display a flashing red light accompanied by rapid beeping. Press either button to stop the beeping. Remove the gel cassette and analyze your results by viewing on one of the transilluminators. The instructor may take a picture of your gel as well, to facilitate quantifying the amount of DNA present in your samples. Page 10

11 4. What did you see from the gel? Draw your results below! Qualitative Testing to Confirm Our Results In the previous steps, we isolated DNA, performed PCR and gel electrophoresis, and visualized DNA confirming the presence of a specific gene that renders the bacteria resistant to a certain antibiotic. Based on these results, we can hypothesize whether our bacteria will survive or die in the presence of any of the three antibiotics we discussed. Please write your hypotheses below: My bacteria will (live/die) in the presence of ampicillin. My bacteria will (live/die) in the presence of chloramphenicol. My bacteria will (live/die) in the presence of kanamycin. Now, we will test your hypotheses. Materials: 15 ml of broth media E. coli culture stock (3-5 ml tube) Pipette and pipette-tips All three antibiotics as solution (Ampicillin, Kanamycin, Chloramphenicol) Bunsen burner Procedure: 1. Spray 70% ethanol on the benchtop surface. Put on your gloves. Obtain your E. coli culture stock (3-5 ml, prepared for you by your instructor), along with the other materials you need. Tighten Page 11

12 the lid on the tube of broth culture, then vortex each tube to suspend the bacteria that might have settled to the bottom of the tube. 2. Ignite the Bunsen burner and work close to it for steps 3-7 below. 3. Portion the 15 ml out into three 5 ml divisions, each in a 15 ml tube. 4. Label the tubes, one each: A, C, and K, for ampicillin, chloramphenicol, and kanamycin. 5. Add 5 μl of the indicated antibiotic into the respective tube: - A = 5 μl of ampicillin - C = 5 μl of chloramphenicol - K = 5 μl of kanamycin 6. Gently invert the tubes a couple of times to mix the antibiotics evenly. 7. Vortex the bacterial stock briefly, then transfer 20 ul of the suspension into each of the 3 tubes from step 5. Incubate for 24 hours at 37 C. 8. After incubation is complete, record your observations. A cloudy broth indicates bacterial growth. This means the antibiotic did not kill the bacteria, demonstrating antibiotic resistance. Observations: Tube A: Tube C: Tube K: Did these observations align with your expectations from the gel electrophoresis procedure? Please explain:. Page 12