Module 2 Lab 2 Bacterial Viruses Week of 2/28-3/4/05 Learning objectives Objectives Explain how bacteriophage infect their hosts and multiply Recognize the bacteriophage infection of bacteria Use PCR to detect the presence of specific viruses Laboratory Learning Objectives overview Hour 1: Watch Lambda bacteriophage video. Hour 2: Set up the bacteriophage plating. Set up the PCR for viral detection. Hour 3: Examine results of antibiotic and detergent bacterial inhibition experiment Readings/ Learning resources Objectives Web resources: http://www.phage.org/ http://www.cdc.gov/ BC1002, Spring semester 2004, Module 2, Lab 2-1
BACTERIOPHAGE EXPERIMENT Bacteriophage T4 infecting a cell Viral plaques The bacteriophage T4 Viruses contain some of the structures and characteristics that are diagnostic of organic life, but they are missing others most notably the biosynthetic machinery necessary for reproduction. Overall, viruses are composed of a single strand of genetic information encased within a protein capsid. In order for a virus to replicate, it must infect a suitable host cell. A bacteriophage is a virus that infects bacteria. Today we will be examining three bacteriophage viruses: T4, F1 and Lambda (λ). All three viruses infect the bacterium E.coli. Each of these three viruses has a distinct replication mode during their infection cycle. The bacteriophage T4 typifies lytic viral life cycles. It exists in an inactive state until one of its extended tail fibers come into contact with the surface of an individual of E. coli. Sensors on the ends of its tail fibers recognize binding sites on the surface of the host's cell, and this triggers the bacteriophage into action. T4 binds to the surface of the host cell and punctures it with its injection tube. Subsequently, T4 injects its own genetic blueprint into the host cell. This genetic information is incorporated into the host cell's normal operation and sets the cell's biosynthetic machinery to work creating replicas of the virus. Once the new virus particles are created within the cell, the cell breaks open (lyses), releasing the new viruses into the media. These released viruses then float about dormant until one comes into contact with a new host cell. The bacteriophage F1 typifies secreted viral life cycles. Like bacteriophage T4, it exists in an inactive state until it contacts a bacterium. The cell surface receptor for bacteriophage F1 is a protrusion on the bacterium that is used by the bacterium to exchange bacterial DNA (the pillus), and the F1 DNA enters the cell through this protrusion. (Bacteriophage F1 can be considered a "venereal disease" for bacteria.) Once inside the cell, the DNA replicates and forms viral particles. Unlike the case for the T4 bacteriophage, the cell doesn't lyse -- instead it oozes bacteriophage particles out into the media. Bacteriophage F1 converts a healthy E. coli bacterium into a factory for producing bacteriophage particles! The bacteriophage λ typifies the lysogenic viral life cycle. Like all other viruses it exists in an inactive state until it contacts a bacterium. Like bacteriophage T4, when it encounters the bacterial cell surface, it injects its DNA into the host cell. However, once the DNA is inside it BC1002, Spring semester 2004, Module 2, Lab 2-2
has a choice: to go lytic or lysogenic. If it goes lytic, it replicates the DNA, makes viral particles and lyses the cell to release the virus. If it goes lysogenic, the DNA inserts into the DNA of the bacterial chromosome, without otherwise affecting the cell, creating a latent, noneffective infection. The bacterium then multiplies and every time it replicates its DNA, it replicates the viral DNA. When conditions get harsh for the bacterium with this latent infection, such that it has a hard time replicating, the viral DNA re-exerts its option to go lytic, whereby it creates new viral particles and lyses the cell. The plaque assay Dilutions of the virus are used to infect a cultured cell layer and covered with agar to restrict bacterial movement. A viral infection of one cell as this cell layer grows releases virus that then affects adjacent cells. These infected cells release virus that affect more adjacent cells, and so on. This results in a region of localized cell inviability (death) and the appearance of plaques (spots on the plates of bacterial lawn growth). The number of plaques directly relates to the number of infectious virus particles applied to the plate, where each plaque results from the chain reaction effect from the initial infection by a single bacteriophage. The different life cycles of the three different phage result in three different plaque morphologies. For bacteriophage T4, with a strictly lytic life cycle, the plaque is sharp and clear. All cells in the vicinity of the original infection are killed. For bacteriophage F1, with a secreted life cycle, the plaque has diffuse edges. No cells are killed by an F1 infection but their ability to replicate has severely impaired. In the center of the plaque, where the first cells were infected, virtually no bacteria have replicated and the plaque is still clear. At the edge of the plaque, where the last bacteria were infected, some bacteria managed to replicate before the infection resulting in a hazy bacterial lawn. For bacteriophage λ, with the lysogenic life cycle, the plaque is turbid. While a good deal of cell killing has resulted from viruses that chose the lytic cell, the lysogenic cells with a latent infection replicate to form a diffuse haze within the plaque formed by the lysis. BC1002, Spring semester 2004, Module 2, Lab 2-3
Plaque Assay Procedure BACTERIOPHAGE IDENTIFICATION When viruses are cultivated for study, they are often grown in cell culture rather than in whole organisms. When cultivated in a layer, the progeny virus of the original infection inhibit the growth of cells in a circle around the site of the original infection, forming a single viral plaque. Each plaque in a layer of cells corresponds to one infectious virus that was originally put on the plate. The size and shape of plaque is indicative of the growth parameters of a virus, and therefore can be used to classify the virus. The number of plaques in a given volume of viral stock solution is a measure of the titer or concentration of virus particles in the viral stock. In this exercise, we will characterize a number of bacteriophage (bacterial viruses) that infect Escherichia coli. We will characterize these bacteriophage on the basis of their plaque morphology over the next two weeks. BACTERIOPHAGE PLATING PROCEDURE OVERVIEW FLOW CHART: BC1002, Spring semester 2004, Module 2, Lab 2-4
BACTERIOPHAGE PLATING PROCEDURE (work individually in lab today) 1. Obtain a microcentrifuge tube containing 0.1 ml of an unknown bacteriophage stock from the instructor. Mark it with your initials and with the words stock solution. 2. Number five sterile microcentrifuge tubes: 1 to 5. 3. Place 45µl of phage dilution buffer into each tube. 4. Remove 5µl of bacteriophage stock from your stock tube, and add it to tube 1. Mix by gently tapping the side of the tube. 5. Make serial dilutions of your bacteriophage. Using a fresh pipette tip, remove 5µl of solution from tube 1 and place it in tube 2. Mix by gently tapping the side of the tube. Remove 5µl of solution from tube 2 and place it in tube 3; mix. Remove 5µl of solution from tube 3 and place it in tube 4; mix. Remove 5µl of solution from tube 4 and place it in tube 5; mix. Use a fresh pipette tip for each transfer for accurate dilutions. 6. Label two microfuge tubes: 4A and 5A. 7. Transfer 20µl of phage solution from microfuge tube 4 to microfuge tube 4A, and mix by gently tapping the tube. Repeat this procedure for tubes 5 and 5A. 8. Add 50µl of plating bacteria to microfuge tubes 4A and 5A. Wait at least 15 minutes before proceeding with step 9. In the meantime, label two Petri dishes containing bacterial nutrient agar with your initials, lab day and time (example TuesAM or MonPM) and label them 4 and 5. Think about why you need to mix plating bacteria with the bactiophage. 9. BE SURE TO READ THIS ENTIRE STEP BEFORE PROCEEDING AS YOU WILL NEED TO WORK QUICKLY TO PREVENT THE AGAROSE FROM COOLING BEFORE YOU POUR IT! After the bacteriophage have been absorbed by the bacteria for at least 15 minutes, (but for less than an hour), the phage are ready to plate. Pipette the contents of microfuge tube 4A into a test tube that contains melted agarose. The test tubes are located in a hot water bath on the front desk. You will need to remove the test tube from the hot water bath, add the contents of tube 4A (by pipetting 35uL two times in a row), and pour the contents BEFORE the agarose COOLS. When it cools, it will become chunky and will not spread evenly. If you get chunks, you ll need to remake tubes 4A and 5A and re-pour them onto smooth plates. Immediately after adding the bacteria/phage to the agarose, pour the contents of the test tube onto the surface of plate 4, and tilt the plate back and forth in order to spread the agar over the surface of the plate. Let the plate sit for at least three minutes to allow the agar solidify (like gelatin, it will solidify as it cools). Repeat this procedure for microfuge tube 5A and plate 5. BC1002, Spring semester 2004, Module 2, Lab 2-5
10. After the agar has solidified, place the plates (upside down, why?) in the 37 C incubator overnight to let the plaques form. The plates will be removed from the incubator, sealed with Parafilm (why?), and stored in the refrigerator (why?) until they can be assayed next week. 11. DO NOT THROW AWAY YOUR DILUTION TUBES YET! You will need them to set up the PCR reactions. PCR Detection of Bacteriophage DNA The polymerase chain reaction (PCR) is a process that results in amplification of a selected region of a DNA molecule. This technique can be used to identify specific DNA sequences with a very high-probability of matching, enabling identification of disease-causing viruses and/or bacteria, a deceased person, or a criminal suspect. Regions of DNA up to about 3000 base-pairs (3 kilobases or kb) can be amplified without difficulty, and longer amplifications of up to 40 kb are possible using modifications to the standard technique. PCR has many applications in the field of molecular biology, such as DNA mapping and DNA sequencing. PCR is also used extensively in specialized areas such as forensic science and clinical diagnosis, where the ability to work with very few starting molecules is particularly useful. In order to use PCR, one must already know the exact sequences which flank (lie on either side of) both ends of a given region of interest in DNA (may be a gene or any sequence). The PCR reaction is carried out by mixing the target DNA molecule, which can be present in extremely small amounts, with nucleotides, two synthetic oligonucleotides primers (one for either side of the region to be amplified), and a thermostable DNA polymerase that is resistant to denaturation by heat treatment. Usually Taq DNA polymerase from the bacterium Thermus aquaticus, which lives in hot springs, is used. The two primers must anneal (stick by means of hydrogen bonding between A-T and G-C nucleotide bases) to the target DNA on either side of the region to be amplified, which means that the sequences of these borders must be known so that the appropriate oligonucleotides can be made. The oligonucleotides prime the synthesis of new complementary polynucleotides (hence the term primers), which are made in the 5 to 3 direction. Because the Taq polymerase is thermostable, the reaction mixture can be heated to 95º C without destroying the enzyme activity (if we carried out the reactions using human DNA polymerase, the enzyme would be denatured when it was heated to 95º C, and would therefore be inactivated so that no DNA could be synthesized). At 95º C, the two DNA strands of the double helix separate from each other. When the mixture is cooled down to about 50-60º C, the primers can anneal to the now single-stranded template DNA. Then, the temperature is raised to the optimum temperature for Taq DNA polymerase (72º C), which makes a new DNA strand by adding new nucleotides to the 3' to end of the annealed primer. BC1002, Spring semester 2004, Module 2, Lab 2-6
When this series of temperature changes are repeated, the new strands detach from the template DNA when the mixture is heated to 95º C. When the mixture is cooled down again, more primers anneal to the template DNA and also to the new strands. Then Taq DNA polymerase carries out a second cycle of DNA synthesis These three steps: 1) strand separation; 2) primer annealing; and 3) polymerization; are repeated 25 to 40 times. During each cycle, the DNA downstream from the bound primers is replicated. The rest of the DNA in the original sample doesn't replicate because there are no primers bound to it for the polymerase to replicate from. The end result is many copies of a DNA fragment bounded on either side by primers used for the reaction and, between them, the original DNA sequence from the source DNA. PCR can continue for 30-40 cycles before the enzyme eventually becomes inactivated or the primers or nucleotides are used up. A single starting molecule can be amplified into tens of millions of identical fragments, representing a few micrograms of DNA. The presence of the fragment can be detected by electrophoresing a portion of the reaction on an agarose gel. PCR Summary Diagram BC1002, Spring semester 2004, Module 2, Lab 2-7
Starting today and continuing next week, we will be using the polymerase chain reaction to identify which unknown bacteriophage was present in your plaques by amplifying a fragment from your unknown bacteriophage and comparing its size with fragments amplified from known cultures of λ, F1, and T4. There are three pairs of primers in the PCR reactions you are setting up this week. One pair of primers binds to λ DNA about 1000 base-pairs apart and amplifies a band of about 1.0 kb (= 1.0 kilobase = 1000 base-pairs). Another pair of primers binds to F1 DNA about 600 base-pairs apart and amplifies a band of about 0.6 kb. The third pair of primers binds to T4 DNA about 300 base-pairs apart and amplifies a band of about 0.3 kb. A band is generated only if the reaction includes the specific bacteriophage DNA. For example, a 1.0 kb band is generated only in the presence of λ DNA in the reaction. Thus, the size of fragment generated (0.3, 0.6 or 1.0 kb) indicates the presence of a specific bacteriophage (T4, F1 or λ, respectively). PCR Setup Procedure (work individually): 1. Write your initials on the side of an empty PCR tube (0.6 ml microcentrifuge tube). 2. Put 5µL of the dilution you made in Tube 1 into the PCR tube. 3. A tube containing the PCR reaction mixture can be found in an ice bucket in your lab room (ask your instructor). This mixture contains Taq polymerase, nucleotides (datp, dctp, dgtp, and dttp), all three pairs of primers, and the appropriate buffers for the reaction. To protect the enzyme from denaturation before the experiment begins, it needs to be kept cold. Without removing the tube from the ice, remove 45µl of enzyme mix with a micropippetor and add it to the PCR tube that contains your phage. 4. Tap the tube on the tabletop to collect the liquid at the bottom of the PCR tube. This will also mix the solution together. 5. Put your tube with the other tubes from your section in the thermal cycler. The thermal cycler is set to hold samples at refrigerator temperatures until all the tubes from both lab sections each day are ready to be run. 6. When all the day's tubes are in the thermal cycler, the thermal cycler will run the program that does the PCR. Basically this is, first, to heat the sample to 95º C for 1 minute (which allow the two DNA strands to separate). Second, the temperature is lowered to 55º C for 1 minute (which allows the primers to bind to the separated DNA strands). Third, the temperature is raised to 72º C (which allows the Taq polymerase to polymerize a new strand generated off the primer). These three steps are then repeated 35 times. 7. Next week we will run a portion of this reaction out on an electrophoretic gel to determine the size of the amplified DNA fragment (if a fragment was generated at all). BC1002, Spring semester 2004, Module 2, Lab 2-8
Completion of Module 2, Lab 1: Analysis of Antimicrobial Agents **Please complete the data tables and questions found on pages 1-12, 1-13, 1-14, and 1-16 in last week s lab handout.** These pages, along with this page, will be due at the beginning of lab next week, 3/7-3/11/05. Procedure (Follow the instructions in last week s lab handout and below): 1. Recover your plate that you seeded last week. Also recover the data sheet in which you indicated which antimicrobial in which quadrant. 2. Measure the diameter of the zone of inhibition for each agent for each bacterial species. If the agent had no effect on bacterial growth, record the size of the zone as 0. 3. Post your results to share with the rest of your lab section. Calculate average values for each. Data table for recording lab section averages of zones of inhibition from antimicrobial agent susceptibility test. Plate/quadrant antibiotic or disinfectant concentration lab section average inhibition zone (mm) a1 a2 a3 a4 d1 d2 d3 d4 Using your data and your lab section average values, please answer the questions on page 1-16 of last week s lab handout. If your data differed greatly from the class values, please speculate on why on a separate piece of paper. BC1002, Spring semester 2004, Module 2, Lab 2-9