Lab 5 - Bacteria Identification Virtual Lab- solved PDF

Preview

Summary

Download Lab 5 - Bacteria Identification Virtual Lab- solved PDF

Description

Virtual Lab Bacterial Identification Virtual

Student Handout

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? Inform me 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? 1. Prepare sample from patient and isolate who bacterial DNA. 2. Make many copies of the desired piece of DNA. 3. Sequence the DNA. 4. Analyze the sequence and identify the bacteria. 3. What is "16S rDNA," and how is it used to identify species of bacteria? It is the DNA used for identifying bacteria. It is the region that codes for a small subunit of the ribosomal RNA or 165 rRNA. Using it to identify bacteria species requires that the sample go through a database of all the known 16S rDNA sequences until a matching sequence can be found.

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? Identify a bacterial sample provided by a clinician.

5. Extracting DNA involves which initial step? Pick up a single colony of the grown bacterial colonies and transfer it to the microcentrifuge tube.

6. What is the wire ring used for?

www.BioInteractive.org

Virtual Lab Bacterial Identification Virtual

Student Handout

Used to pick up a single colony of the grown bacterial colonies and transfer it to the microcentrifuge tube.

7. Why are the proteolytic enzymes necessary? Extracting DNA from bacteria involves dissolving the cell wall using a digestive buffer. This buffer contains proteolytic enzymes which eat away at the cell wall oer an extended period of time.

8. Why do you then need to inactivate the proteolytic enzymes and how do you do it? We will use other enzymes which is why we have to inactivate the proteolytic enzymes. To do so, the sample is placed in a heated water bath at 100 degrees Celsius. 9. After removing the enzymes, why do you spin the sample in the centrifuge? The next step is to handle the cellular debris which is why the sample is spun in the centrifuge.

10. a. What is the pellet?

The pellet is the solid at the bottom of the sample tube that use to be the cellular debris. b. What is the supernatant?

The liquid. c. Where is the DNA? Contained 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 (PCR) A method for creating a large number of copies of a specific piece of DNA.

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.)

www.BioInteractive.org

Virtual Lab Bacterial Identification Virtual

Student Handout

Add the Master Mix and answer the following questions: 13. What does the Master Mix contain? it contains water, which acts as a buffer to keep the mixture at a correct pH for the PCR chain to work properly. Also huge quantities of nucleotides; adenine, cytosine, guanine, and thymine; and oligonucleotide DNA primers that help to bind the 16s rDNA area with the goal of starting the replication process. Finally, a stable heated DNA polymerase is required to copy DNA. 14. What are primers? Why is a primer added? Primers, small pieces of DNA for binding sequences, allow two copies of DNA to overlap. 15. Once the primers bind, what occurs next? The 16s rDNA is copied from the bacteria sample. 16. What does "highly conserved" mean? Parts of the gene are similar even in different types of species. 17. Why are highly conserved regions important in this lab? All inclusive groundworks help tie the profoundly rationed territories of the DNA, with the goal that they can be utilized to duplicate DNA from a huge number of animal types 18. What does "highly variable" mean? Parts of gene are extremely different in different species 19. Why are highly variable regions important in this lab?

www.BioInteractive.org

Virtual Lab Bacterial Identification Virtual

Student Handout

Used in identification. 20. What is missing in the negative control tube? 16s rDNA solution. 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. 22. List each step of a PCR cycle, the temperature, and the duration (time). a. Melt at 95 C for 30 seconds b. Anneal at 60 C for 30 seconds c. Extend at 72 C for 45 seconds

23. Describe what happens during each of the steps in one or two sentences. a. Within the melting step the two chains of DNA must separate by heating the tube with the PCR mixture b. Within the anneal step, a single stranded DNA is bound by primers c. Extending step helps the DNA polymerase extend the copied DNA strand with raised temperatures. 24. After eight cycles, how many copies of the desired DNA have been synthesized? 240.

www.BioInteractive.org

Virtual Lab Bacterial Identification Virtual

Student Handout

25. After 30 cycles? 1 million.

PART 3: PCR PURIFICATION 26. Approximately how long is the 16s rDNA (bp)? About 1500 bp.

27. Why would it be useful to run an electrophoresis gel at this point? To confirm that the PCR reaction worked. 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. Negative control: this would contain water and should have no other products unless the water was contaminated. b. Positive control: this would contain PCR product of a known DNA sequence which would serve to make sure the PCR worked. c. Last lane: contains the sample. 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? Should contain many pieces of 1500bp long rDNA with a small amount of longer DNA strands which serve as contaminants.

PART 4: SEQUENCING PREPARATION Click on "Learn about cycle sequencing before proceeding."

www.BioInteractive.org

Virtual Lab Bacterial Identification Virtual

Student Handout

30. Read the first two paragraphs and list the steps in cycle sequencing in the space provided. 1. Utilize a thermocycler to make numerous duplicates of the objective DNA. 2. Ending the replication cycle indiscriminately puts in the process to guarantee the duplicates are various lengths comes straightaway, for the most part because of making the duplicates. 3. Use electrophoresis gel to isolate the pieces dependent on measured Compile the succession for correlative DNA

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. The green and blue tubes both sequencing brew which consist of buffers, primers, 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? The point of the second PCR is to deliver numerous duplicates of different lengths instead of indistinguishable duplicates of DNA. 33. Where do scientists obtain primers to be used in PCR and in this technique? Through commercial sources. 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 blend of DNA bits of different lengths, beginning with a similar preliminary however finishing with an alternate nucleotide labeled with a fluorescent marker. 35. What is the purpose of gel electrophoresis? Method used to separate molecules based on size difference.

36. How do DNA molecules move in relation to charge? Why? DNA particles are adversely charged and as such will in general travel through the cylinder toward the

www.BioInteractive.org

Virtual Lab Bacterial Identification Virtual

Student Handout

decidedly charged needle end. More modest pieces move quicker than the enormous ones. 37. What is the purpose of the laser beam in determining a DNA sequence? To energize the fluorescent markers permitting optical identifiers to distinguish the fluorescence tone.

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 of interest bears a significant degree of similarity to another known sequence. 39. What is "homology"? Similarity between two sequences.

40. What is BLAST and how is it used? Basic Local Alignment Search Tool used for comparing sequence information in proteins or DNA by utilizing all available public sequence databases. 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. It is the numerical score assigned by BLAST while the bits are the measure of information. 43. What does an E-value of 3 or less represent? Represents a meaningful match that is not due to chance that is considered an acceptable match.

www.BioInteractive.org

Virtual Lab Bacterial Identification Virtual

Student Handout

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 ensilage.

45. Write a brief description of this bacterium in the space provided. Bartonella henselae are pathogenic to people. There are different species and they are generally sent through a vector or from a creature supply, for example, felines on account of feline scratch illness. Side effects of the fever regular show in swollen lymph organs, potential skin injuries, conceivably fever and weakness among different indications. 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

C

Pseudomonas aeruginosa

Generally found in soil, water and other regular habitats adept at filling in irregular sources like cleanser buildup When an individual is solid, there's not normally a very remarkable wellbeing danger

D

Salmonella typhimurium

Normally found in the intestinal lot of well evolved creatures, birds and reptiles. The most extreme ailment brought about by these microbes is typhoid fever. A typical hotspot for these microorganisms is uncooked eggs.

F

Yersina enterocolitica

www.BioInteractive.org

These bacteria caused the plague carried by urban and rural rats, via their fleas which will even settle for a human host can potentially be contagious by air as

Virtual Lab Bacterial Identification Virtual well.

www.BioInteractive.org

Student Handout...