Bacterial Identification

Virtual Lab Bacterial Identification Virtual Student Handout INTRODUCTION 1. What is the overall purpose of this virtual lab? Learn about the science and techniques used to identify different types of bacteria based on their DNA sequence. 2. What are the four basic steps involved in this bacterial identification lab? Prepare a sample from patient and isolate whole bacterial DNA. Make many copies of the desired piece of DNA. Sequence the DNA. Analyze the sequence and identify the bacteria. 3. What is "16S rDNA," and how is it used to identify species of bacteria? The piece of DNA used for identifying bacteria is the region that codes for a small subunit of the rRNA. Click to Enter the Lab. (Click the window on the left-hand side of the screen to enter the lab.) As you enter the lab, follow the instructions in the lab (left-hand window). Using the information in the Notebook window on the right, answer the following questions. PART 1: SAMPLE PREPARATION 4. As the pathology lab technician, what is your task in this virtual lab? Your task is to identify a bacterial sample received from a clinician. 5. Extracting DNA involves which initial step? Picking up a single colony and drop it into a microcentrifuge tube. 6. What is the wire ring used for? For scooping up bacteria. Virtual Lab Bacterial Identification Virtual Student Handout 7. Why are the proteolytic enzymes necessary? To lyse the bacteria cell wall. 8. Why do you then need to inactivate the proteolytic enzymes and how do you do it? Because we need to introduce a new enzyme. The proteolytic can be denatured by heating the sample in a water bath at 100 °C. 9. After removing the enzymes, why do you spin the sample in the centrifuge? To separate the DNA from other cellular debris. 10. a. What is the pellet? Cellular debris b. What is the supernatant? Liquid suspended above the pellet c. Where is the DNA? In the supernatant PART 2: PCR AMPLIFICATION Go on to Part 2 and work through the PCR steps. Be sure to read the information in the notebook, including “What is PCR?” 11. What does “PCR” stand for and what is the purpose of PCR? Polymerase Chain Reaction. Use for multiplying DNA copies. 12. Summarize the process of PCR in a diagram. Include all the steps, labeled and in the right order. (If you are completing this handout online, draw the diagram on a piece of paper, take a photo, save the image as a PDF, and upload it in the space below.) Virtual Lab Bacterial Identification Virtual Student Handout Virtual Lab Bacterial Identification Virtual Student Handout Add the Master Mix and answer the following questions: 13. What does the Master Mix contain? Water, a buffer, large quantities of the ATCG, large quantities of oligonucleotide DNA primers, and a heat-stable DNA polymerase. 14. What are primers? Why is a primer added? To bind to the 16S rDNA region to initiate the replication process. 15. Once the primers bind, what occurs next? The DNA polymerase attaches to it and begin adding ATCG in the 3’ to 5’ direction. 16. What does "highly conserved" mean? Part of genes that are extremely similar in related species. 17. Why are highly conserved regions important in this lab? So that the primers bind to the highly conserved regions of genes so that they can be used to copy DNA from a variety of species of bacteria. 18. What does "highly variable" mean? Section of genome that adapt to each bacteria environment. Changing as the bacteria needs changes. 19. Why are highly variable regions important in this lab? For identifying bacteria. 20. What is missing in the negative control tube? Sample DNA. 21. What is present in the positive control tube that is not in the negative control tube? Positive control DNA. Now run the PCR. Be sure to watch the virtual lab animation before proceeding to the questions . Virtual Lab Bacterial Identification Virtual Student Handout 22. List each step of a PCR cycle, the temperature, and the duration (time). a.  Melt: 95°C 30 seconds b. Anneal: 60°C 30 seconds c. Extend: 72°C 45 seconds 23. Describe what happens during each of the steps in one or two sentences. a. Separate each strand for the DNA by breaking the hydrogen bonds. b. Allowing the primer to attach to each strand. c. Allowing DNA polymerase to elongate each strand. 24. After eight cycles, how many copies of the desired DNA have been synthesized? 256 copies. 25. After 30 cycles? Approximately 1 million copies. PART 3: PCR PURIFICATION 26. Approximately how long is the 16s rDNA (bp)? 1500 base pairs. 27. Why would it be useful to run an electrophoresis gel at this point? Virtual Lab Bacterial Identification Virtual Student Handout To confirm that the PCR works. 28. If you were to run a gel, it would have three lanes. What would each lane contain, and what would you see in each lane after running the gel? a. Water. Should not be any product unless contamination. b. PCR product of known DNA sequence. Moving through the lane. c. Sample. Moving through the lane but different from the PCR product’s location. 29. The gel is not run in this virtual lab. In order to purify the PCR product, you use a microconcentrator column. (Proceed through the virtual lab steps.) What should the final collection tube contain? Many pieces of 1,500 bp-long 16S rDNA. PART 4: SEQUENCING PREPARATION Click on "Learn about cycle sequencing before proceeding." 30. Read the first two paragraphs and list the steps in cycle sequencing in the space provided. First step is to use a thermocycler to create many copies of the target FNA but terminate the replication process at random places, so the copies are all partial sequences with different lengths. The reaction mixture contains normal DNA as well as some special Dideoxynucleosides that are tagged with fluorescent markers. The fluorescent markers differ for each base and are designed to fluoresce with different colors. DiDNA can substitute for normal DNA during replication, but if it happens the chain can no longer be extended. DiDNA are thus called terminators. Click to go back to Part 4. 31. What do the green and blue tubes contain? Describe the “sequencing brew” to which you added your purified PCR. Green and blue tubes contains a sequencing brew consisting of buffers, primer a different one for each tube, DNA polymerases, nucleotides, and fluorescence-tagged terminators in suitable proportions. 32. The purpose of the second PCR is not to create identical copies like the first PCR you ran. What is the purpose of this PCR? Virtual Lab Bacterial Identification Virtual Student Handout To produce many copies of variable length, 33. Where do scientists obtain primers to be used in PCR and in this technique? Commercial source. Watch the virtual lab animation before proceeding to Part 5. PART 5: DNA SEQUENCING 34. What is the final PCR product, the stuff contained in your 12 tubes? A mix of DNA pieces of variable length. 35. What is the purpose of gel electrophoresis? To separate molecules based on differences in size. 36. How do DNA molecules move in relation to charge? Why? Because the DNA molecules are negatively charge, they would move through the tube toward the positively charged syringe end. 37. What is the purpose of the laser beam in determining a DNA sequence? Collect the information from the tubes by light refraction. Be sure to watch the virtual lab animation before proceeding to Part 6. PART 6: DNA SEQUENCE ANALYSIS Click on "Learn about the science behind sequence matching." 38. What is the ultimate goal of the sequence matching analysis? To determine whether the new sequence bears a significant degree of similarity (or homology) to another known sequence. 39. What is "homology"? The state of having the same or similar relation, relative position, or structure. Virtual Lab Bacterial Identification Virtual Student Handout 40. What is BLAST and how is it used? Basic Local Alignment Search Tool offer a good combination of speed, sensitivity flexibility, and statistical rigorousness for matching rDNA sequence. 41. What’s a major assumption when drawing evolutionary relationships between organisms based on DNA sequences? The number of positions that differ in the nucleotide sequence is proportional to the time elapsed since the two species formed their own lines of descent from a common predecessor. Click to go back to Part 6 and click on "Learn more about BLAST search results." 42. Explain what the "Score (bits)" means on an actual BLAST search result. Measure of information. Two for each matching pair. The higher the score, the better is the match. 43. What does an E-value of 3 or less represent? An acceptable match. Click to go back to Part 6 and proceed through the instructions in the right-hand notebook window. ● Hints: "Ctrl A" will select all the data in the pop-up window, "Ctrl C" will copy it, and "Ctrl V" will paste it into the NCBI website (large yellow box at the top of the BLAST search page). ● Follow the steps listed on the page and be patient. BLAST data can take a while to search. ● When the BLAST results appear, scroll down below the color key to the significant alignments, and then go back to the virtual lab window (left) and follow the instructions. 44. What is the scientific name of the bacterium you sequenced? Bartonella henselae 45. Write a brief description of this bacterium in the space provided. Mostly pathogenic to human. Could cause disease via vector, that could result in: Oroya fever, trench fever, cat scratch disease. Some symptoms are swelling of lymph glands, skin lesion at the site of inoculation, fever, fatigue, and others. People with weak immune system are more susceptible and can Virtual Lab Bacterial Identification Virtual Student Handout develop different disease such as angiomatosis. After completing Sample A, perform DNA sequence analysis on three of the other five samples. 46. Write in the letter of the samples you choose, the scientific name of the bacterium (after doing a BLAST search), and a brief description of each. Sample Letter Bacteria Scientific Name Brief Description B Escherichia coli Probably the most famous bacterium, E. coli is one of the most common inhabitants of the intestinal tract. Generally, they are not considered pathogenic, but they can cause disease under certain conditions. There are pathogenic strains that can cause diarrhea or other serious conditions. C Pseudomonas aeruginosa Pseudomonads are commonly found in soil, water, and other such natural environments. To healthy individuals, these bacteria do not generally constitute a health threat. They have been found to be remarkably adept at growth by using most unusual sources of nutrients such as soap residues or glue. In hospitals, P. aeruginosa has been known to cause problems in debilitated patients and is a common cause of disease in children with cystic fibrosis. These persistent creatures have been found in unlikely places such as air vents, water hoses, and even in detergent holding tanks in the very machines that are used to clean medical instruments. D Salmonella typhimurium Many strains of Salmonella are potentially pathogenic. They are commonly found in the intestinal tract of mammals, birds, and reptiles. Under some conditions, they can lead to contamination of food. The most severe illness caused by any Salmonella is typhoid fever (pathogen: Salmonella typhi). Other strains, of which S. typhimurium is one, cause less serious gastrointestinal diseases, collectively known as Salmonellosis. The bacteria invade the cells of the intestinal tract of the host, multiply, and sometimes escape to the bloodstream and the lymphatic system. Symptoms include fever, nausea, abdominal pain, and Virtual Lab Bacterial Identification Virtual Student Handout diarrhea. Nowadays, uncooked eggs are a particularly common source of salmonellosis. Soft-boiled eggs, incompletely cooked yolk in fried eggs, or use of raw or incompletely cooked eggs in sauces and desserts can all present a risk.