Dev Poduval Bacterial Identification Lab Worksheet

To use this document, first read the Instructions and FAQs. This document is licensed by the Howard Hughes Medical Institute under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International license. No rights are granted to use HHMI’s or BioInteractive’s names or logos independent from this document or in any derivative works. Using this document, you agree to use this document in accordance with these terms. INTRODUCTION Go to https://www.hhmi.org/biointeractive/explore-virtual-labs. Scroll down and click on “The Bacterial Identification Virtual Lab.” Maximize the screen if you wish. Answer the following questions in the spaces provided. 1. What is the overall purpose of this virtual lab? To learn how scientists identify bacteria by analyzing their DNA. 2. What are the four basic steps involved in this bacterial identification lab? Collect the sample and pull out its DNA, Amplify the DNA section you care about, Determine the exact DNA sequence, Compare that sequence to known bacterial databases. 3. What is "16S rDNA," and how is it used to identify species of bacteria? It’s a ribosomal gene found in all bacteria, and its sequence can be matched to known 16s sequences to pinpoint the species. 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? Figure out which bacterium is present in the unknown sample. 5. Extracting DNA involves which initial step? The cell wall has to be broken open using a digestive buffer. 6. What is the wire ring used for? To scoop up a single bacterial colony from the plate. www.BioInteractive.org 7. Why are the proteolytic enzymes necessary? They break apart the cell structures so the DNA can be released. 8. Why do you then need to inactivate the proteolytic enzymes and how do you do it? Because the next steps require them to be inactive, so you heat the tube to boil them and stop their activity. 9. After removing the enzymes, why do you spin the sample in the centrifuge? Spinning forces all the leftover cell fragments into a pellet at the tube’s bottom. 10. a. What is the pellet? The clump of solid cell debris. b. What is the supernatant? The clear liquid that sits above the pellet after centrifuging. c. Where is the DNA? Suspended in the supernatant, not in the pellet. 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, a method that massively multiplies a specific DNA region. 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.) Add the Master Mix and answer the following questions: www.BioInteractive.org 13. What does the Master Mix contain? It holds the nucleotides, buffer, salts, magnesium, and everything needed for PCR to run. 14. What are primers? Why is a primer added? They’re short DNA starters that attach to the target region so copying can begin. 15. Once the primers bind, what occurs next? The polymerase starts building new DNA strands in the 5’-> 3’ direction. 16. What does "highly conserved" mean? A region that barely changes across different species. 17. Why are highly conserved regions important in this lab? Because universal primers can latch onto them in almost any bacterial DNA. 18. What does "highly variable" mean? They provide the differences needed to tell species apart. 19. Why are highly variable regions important in this lab? They provide the differences needed to tell species apart. 20. What is missing in the negative control tube? The bacterial DNA only contains water. 21. What is present in the positive control tube that is not in the negative control tube? Now run the PCR. Be sure to watch the virtual lab animation before proceeding to the questions. 22. List each step of a PCR cycle, the temperature, and the duration (time). a. 95 degrees Celsius for 30 sec: melting www.BioInteractive.org b. 60 degrees Celsius for 30 sec: annealing c. 72 degrees Celsius for 45 sec: extension 23. Describe what happens during each of the steps in one or two sentences. a. Heat separates the two DNA strands. (during melt: 95 degrees celsius for 30 sec) b. Cooling lets primers attach to the single-stranded DNA. (during annealing: 60 degrees celsius for 30 sec) c. The polymerase builds new copies of the DNA. (during extension: 72 degrees Celsius for 45 sec) 24. After eight cycles, how many copies of the desired DNA have been synthesized? Around 240 copies. 25. After 30 cycles? Around one billion. PART 3: PCR PURIFICATION 26. Approximately how long is the 16s rDNA (bp)? About 1.5 kb (1500 bases). 27. Why would it be useful to run an electrophoresis gel at this point? To verify that the PCR actually produced the expected DNA band. www.BioInteractive.org 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. b. c. 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? Mostly clean 1500-bp 16s rDNA fragments. 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. 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. All the reagents for sequencing, including primers, buffers, polymerase, nucleotides, and fluorescent terminators. 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? To create DNA pieces of many different lengths for sequencing. 33. Where do scientists obtain primers to be used in PCR and in this technique? They use custom-designed primers that bind at specific spots and include fluorescent tags. www.BioInteractive.org 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 bundle of DNA fragments that vary in length. 35. What is the purpose of gel electrophoresis? To sort DNA molecules based on size. 36. How do DNA molecules move in relation to charge? Why? They travel toward the positive end because DNA naturally carries a negative charge. 37. What is the purpose of the laser beam in determining a DNA sequence? 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 see which known organism your DNA sequence matches most closely. 39. What is "homology"? The degree of similarity between two sequences. 40. What is BLAST and how is it used? A database tool that compares your DNA sequence to millions of known ones. 41. What’s a major assumption when drawing evolutionary relationships between organisms based on DNA sequences? More sequence differences mean the species split from a common ancestor longer ago. www.BioInteractive.org 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. The score shows how strong the match is; bits measure the informational value of that score. 43. What does an E-value of 3 or less represent? It’s considered a solid, reliable 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. Bartonella henselae is the bacteria that causes cat-scratch disease, usually spread from cats to humans. 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 www.BioInteractive.org Brief Description www.BioInteractive.org