AP_Bio_Lab_8_Bacterial_Transformation

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Indiana University, Bloomington *

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L112

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Biology

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Apr 25, 2024

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AP Bio Lab 8 Bacterial Transformation Modified Lab Report I am modifying the requirements for this lab. You DO NOT have to turn in a formal lab report. Please complete the following lab sheet as an informal lab report. Pre-Lab Activity To prepare for the following lab review the relevant information on bacterial transformation in the content, sidebar, and in your textbook. View the DNA Transformation videos: https://gpb.pbslearningmedia.org/resource/biot11.sci.life.gen.transbact/transforming-bacteria/ Answer the following questions: 1. What is DNA transformation? The act of introducing foreign DNA into a cell and allowing it to integrate and express itself inside the cell is known as DNA transformation. In genetic engineering and biotechnology, it is frequently used to alter organisms for various goals, such as generating helpful proteins or researching gene function. 2. What is the purpose of using “heat shock” in transformation? “Heat shock” is a technique used in transformation to momentarily increase the permeability of the cell membrane, making it easier for foreign DNA to enter the cell. 3. Describe the process researchers use to make a bacterial cell capable of taking up new DNA. Bacterial transformation is a technique used by researchers to enable a bacterial cell to absorb additional DNA. This gives the cells a short heat shock to enable the foreign DNA to enter cells after treating them with calcium chloride to increase the permeability of their cell membrane. Procedure This is a very complex lab originally designed to be conducted in the classroom. However, because of the complexity of the lab, you will conduct a dry lab based on hypothetical data. Begin by reading the procedure included in the lab manual on pages 102-105. You will now go through a virtual version of this lab and “collect” results. Go to the Key Concepts I section of the virtual Bacterial Transformation Lab. In this section you will review the basic concepts needed to understand what is going on in the lab. 1. Watch the animation about “Bacterial Transformation” and read the information on this page. Once completed select “Continue to Bacterial Colonies” to advance. 2. Read the information on “Bacterial Colonies.” Once completed, select any E.coli colony in the petri dish to learn more about his bacteria. 3. Read the information on “ E. coli Bacteria.” Once completed, select “Continue to Plasmids” to advance. 4. Read the information on “Plasmids.” Once completed, select “Continue to Competent Cells” to advance. 5. Read the information on “Competent Cells.” Once completed, you have completed reviewing the key concepts for this lab. You are now ready to advance to the virtual lab. Select “Continue to Design the Experiment I.”

“Design of the Experiment I” section . Complete the following: 1. If you were doing this lab live, it would be imperative that you follow sterile laboratory procedure both for the function of the investigation and the safety of the investigator. Read through the sterile laboratory procedures. Once completed, select “Continue to Transformation Procedure” to advance. 2. Read the introduction to “Transformation Procedure.” Carefully review the graphic and click on each step to see what is happening at the cellular level. 3. Review the process as a whole at the cellular level by viewing the animation. 4. Check for understanding . Select “Place the Stages of Transformation in Order.” Follow the instructions for this self-check and then indicate the correct order of events for bacterial transformation at the cellular level on this lab sheet by numbering the following images. Data Once you have completed the self-check, ordered the images on your lab sheet, and summarized the procedure, click on “ Continue to Analysis of Results I ” to advance. 1. Read the page on “Analysis of Results I” and review the graphic. Once completed, click on “continue to Label the Results of Your Experiment” to advance. 2. Check for understanding. Complete the self-check and add the correct labels to section in yellow for the data table below: Table 1: Ideal Bacterial Growth Plates for Bacterial Transformation of E. Coli with Ampicillin Resistant Plasmid TYPE OF BACTERIA G R O W T H M E D I A Analysis and Discussion Analysis Questions ~ Use the plates in Table 1 to answer the following questions: 1. On which of the plates would you expect to find bacteria most like the original non-transformed E. coli colonies you initially observed? Why? Bacteria most like the original non-transformed E.coli colonies observed initially on plate 1. This is because that plate does not contain ampicillin and a negative

amount of the ampr cells. This plate also allows most things to grow. 2. If there are any genetically transformed bacterial cells, on which plate(s) would they most likely be located? Again, why? Plates 2 and 4 because these are the plates that contain plasmid which is what lets the transformation occur. 3. Which plates should be compared to determine if any genetic transformation has occurred? Why? Plates 3 and 4 should be compared to determine if a genetic transformation has occurred. The +ampr had plasmid while the -ampr did not. Use the results shown below to answer the next set of questions. Remember that LB is the bacteria food, amp is the ampicillin antibiotic, and + means that those bacteria were exposed to the plasmid. LB - plate Lawn of bacteria (covered in bacteria) – do not glow LB / amp - plate No bacteria at all LB + plate Lawn of bacteria (covered in bacteria) – no visible glowing LB / amp + plate Scattered colonies of bacteria – these glow For the claims below, tell me which TWO plates we would need to compare TO EACH OTHER to illustrate the claim. Explain…. how do we know? 1. The plasmid had the gene for ampicillin resistance and it worked as intended. To illustrate the claim that the plasmid had the gene for ampicillin resistance and it worked as intended, you would need to compare the LB + plate (bacteria exposed to the plasmid without ampicillin) with LB/amp + plate (bacteria exposed to the plasmid with ampicillin). If ampicillin resistance is successful you would observe a lawn of bacteria on the LB +plate but scattered colonies on the LB/amp + plate due to the antibiotic selection. 2. The plasmid had the lux gene that caused the transformed bacteria to glow To illustrate the claim that the plasmid had the lux gene that caused the transformed bacteria to grow you would need to compare the LB + plate (bacteria exposed to the plasmid without ampicillin) with the LB/ amp + plate (bacteria exposed to the plasmid with ampicillin). If the lux gene is present and functional, you would observe glowing on the LB / amp+ plate but not on the LB + plate. 3. Very few of the bacteria in the + tube actually transformed. To illustrate the claim that very few of the bacteria in the + tube actually transformed, you would need to compare the LB + plate (bacteria exposed to the plasmid without ampicillin) with the LB / amp + plate (bacteria exposed to the plasmid with ampicillin). If very few bacteria transformed, you would observe a lawn of bacteria on the LB + plate but only scattered colonies on the LB / amp + plate.

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