Bacterial Transformation with Green Fluorescent Protein. pgfp Version. Table of Contents Fall 2012

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1 Bacterial Transformation with Green Fluorescent Protein pgfp Version Table of Contents Bacterial Transformation Introduction..1 Laboratory Exercise...3 Important Laboratory Practices 3 Protocol Worksheet: Bacterial Transformation Acknowledgements..10

2 Introduction to Bacterial Transformation Transformation is a process of transferring genetic information from one organism to another. In bacteria, a small circular piece of DNA known as a plasmid (Table 1), transfers genetic information between bacteria, allowing these microbes to gain antibiotic resistance and adapt to new environments. This natural process can be modified by humans to increase our quality of life. In agriculture, genes are added to help plants survive difficult climatic conditions, insect damage and increase their nutrients. Toxic chemical spills are often controlled by transformed bacteria. Currently, many diabetics rely on insulin made from bacteria transformed with the human insulin gene. Scientists use transformation as a tool to work on ways to treat other human diseases and conditions. Table 1: Illustration of a bacterial cell with chromosome and plasmids Symbol Bacterial structure Illustration of E. coli Plasmid containing a few genes Circular bacterial chromosome In this lab, you will be using non-pathogenic E. coli bacteria and pgfp, a plasmid modified with two genes. The pgfp plasmid contains the genetic codes for (see Table 2): 1. a green fluorescent protein (GFP) from the bioluminescent jellyfish, Aequorea victoria 2. ampicillin resistance (amp) If pgfp transformation is successful and the bacteria are growing, the colonies will appear green under natural light fluorescent, neon green under UV light. These green bacteria must contain the plasmid with the GFP. For this reason, the green fluorescent protein (GFP) gene is often used as a reporter gene to identify expression of other genes of interest. Table 2: pgfp plasmid and its two important genes. Type of gene Ampicillin resistance gene forming betalactamase, which inactivates ampicillin in media pgfp plasmid with inserted genes GFP% gfp gene produces the green fluorescent protein (GFP) Amp R% 1

3 In order to successfully transform bacteria, you will need to add CaCl 2, transformation solution (TS), to neutralize both the bacterial cell wall and membrane charges, then, quickly shock them with a temperature change in order for them to uptake the pgfp plasmid. After the stressful event, you will provide nutritious broth to restart their growth. Deviating from the protocol listed below may decrease your success in obtaining transformants. Will the untransformed bacteria appear neon green under a UV lamp? Will transformed bacteria fluoresce under a UV lamp? List your predictions below. Before beginning the transformation, observe a plate of E. coli and a vial of pgfp plasmid under a UV lamp. Then, view your transformed colonies once you complete the protocol below. Explain your results. Predictions & Actual Results Item Prediction View with UV Lamp Explanation of Results E. coli growing in petri dish Vial of pgfp plasmid Transformed E. coli in petri dish 2

4 Laboratory Exercise The protocol outlined below describes a procedure for adding plasmid DNA to a bacterial cell. You will cool them quickly and briefly heat the cells to move the plasmid DNA into the cells. Then, you will grow the cells on a petri dish containing LB agar and antibiotics. You will look for the development of green colored colonies of bacteria. Objectives - student should be able to: 1. Understand what a bacterial plasmid is and how it is used in biotechnology. 2. Understand how genes are used to make protein in a cell. a. Add reagents to the bottom of the reaction tube, not to its side. b. Add each additional reagent directly into previously added reagent. c. Do not pipet up and down to mix, as this introduces error. d. Make sure contents are all settled into the bottom of the tube and not on the side or cap of tube. A quick spin may be needed to bring contents down. Important Laboratory Practices a. Pipet slowly to prevent contaminating the pipette barrel. b. Change pipette tips between each delivery. c. Change the tip even if it is the same reagent being delivered between tubes. Change tip every time the pipette is used Keep reagents on ice. Check the box next to each step as you complete it. 3

5 Place a check mark in the box as you complete each step. Protocol 1. Sterilize lab surfaces and wash hands before beginning the lab. 2. Obtain two empty 1.5mL microfuge tubes from your instructor. Using a permanent marker, label one tube +DNA and the other tube DNA. Label each tube twice, on the lid and on the side. Place these tubes into a Styrofoam cup containing crushed ice. 2. Add 250µL of Transformation Solution (TS) to each tube. If using a P-200 micropipettor, set the dial to 125µL and transfer 250µL (125µL, 2 times) Note: TS contains calcium chloride (CaCl 2 ), which helps neutralize both the bacterial cell wall membrane and DNA charges. Keep your tubes on ice. 4. Obtain a starter plate of E. coli. Observe the colonies growing on it and note what you see. 250µL TS DNA Wear safety glasses while using the UV lamp. UV#Light# 5. With a sterile inoculation loop, pick up one bacterial colony from the starter plate. Dip and swirl the loop into the +DNA tube to evenly disperse the colony in the solution and release it from the loop. With the cap closed, flick the tube with your finger to mix. Use a new loop to repeat the process for the - DNA tube. Return tubes to ice. 6. Wearing safety glasses, observe the contents of a vial of pgfp under a UV lamp. 7. With a P-20 micropipettor, transfer 10µL of the pgfp plasmid into your tube labeled +DNA only. DO NOT add plasmid to the DNA tube. UV#Light# Close the cap and flick the tube to mix the plasmid with the contents of this tube. Keep tubes on ice. 250µL pgfp 4

6 8. Incubate both tubes on ice for 10 minutes, making sure the tubes are in contact with the ice. DNA 9. While you re waiting, pick up these 3 plates: 1 LB and 2 LB/amp On the outer edge on bottom of the plate, write +DNA onto one of the LB/amp plates. Write DNA on the LB plate and the second LB/amp plate. Also place your team initial or symbol on the bottom of each plate. 10. Heat shock your bacteria by transferring both tubes to a foam rack and placing them into a water bath set at 42 C for 50 seconds. 10 minutes on ice Plate Name LB DNA LB/amp DNA LB/amp +DNA DNA Make sure the tubes are pushed down as far as they can go in the rack to contact the hot water. DNA DNA After 50 seconds, quickly place both tubes on ice for another 2 minutes. It is VERY important to watch the time and speed of the transfers. 11. Return your tubes to a tube rack now resting on your lab bench. Water&Bath& 42 C%/%50%seconds% 2%min% Add 250µL of LB broth to each of the tubes. If using a P-200 micropipettor, set the dial to 125µL and transfer 250µL (125µL, 2 times) 250µL LB Remember to change the tips between the tubes 12. Close the tubes. Mix each tube by flicking it several times with your finger. Incubate the tubes for at least 10 minutes at room temperature. This process will allow the transformed bacteria to recover and provide nutrients for their growth. 13. Obtain your three labeled plates. Transfer 100µL of the appropriate solution to each labeled plate. Remember to change tips between each transfer 10 minutes at room temperature Plate Name LB DNA LB/amp DNA LB/amp +DNA Add100µL of cell solution 5

7 14. With a new sterile loop for each plate, spread the liquid you just transferred onto each of the plates. Evenly cover as much of the plate as possible. Discard used tips into a waste container with disinfectant. Allow bacteria to saturate into the agar plate for a few minutes before the next step. 15. Invert your three plates. Then stack and tape them together. Place plates into an incubator oven set at 37 C until the next day or when colonies are visible. 16. Decontaminate all lab surfaces with dilute disinfectant and wash hands following the lab 6

8 Name Date Period Worksheet: Bacterial Transformation Lab Predictions Will the untransformed bacteria, pgfp plasmid, and transformed bacteria all fluoresce green? Before viewing these substances with a UV lamp, list your prediction on whether they will fluoresce green. Then, view them under a UV lamp and provide an explanation of your results. 1. Predictions & Results Item Prediction With UV lamp Explanation E. coli colony Vial of pgfp plasmid Transformed E. coli colony 2. Explain the purpose of these processes or substances during transformation. Process or Purpose Substance a. LB agar b. Prevents growth of untransformed bacteria on LB/amp plates. c. Calcium chloride d. Heat shock 3. Describe 2 differences and 2 similarities between these Bacteria. Condition - pgfp DNA bacteria + pgfp DNA bacteria Difference Similarity 7

9 Name Date Period 4. Before transforming your bacteria, list your predictions below for each of these petri dishes and their contents. Then, describe your results following transformation. Contents LB -DNA LB/amp -DNA LB/amp +DNA Predictions* Answers vary Answers vary Answers vary Illustration of Results Description of Results *Possible responses for Predictions - Growth or no growth, fluorescence or no fluorescence under UV light, number of colonies, etc.) 5. Compare your predictions with your actual lab results. Describe how close your predictions were to your actual results and explain possible reasons for any differences. Answers may vary. If responses are different from their results, students may not understand the process and need further review. 6. Explain what may have occurred to produce these results. ( = colony) Contents LB -DNA LB/amp -DNA LB/amp +DNA Illustration of Results Description of Results Possible explanation for results 8

10 Name Date Period 7. If growth appeared on the LB/amp +DNA plate, would these bacteria a. be transformed? Explain. b. fluoresce under UV light? Why or why not? 8. Provide an example of how transformation can be beneficial and an example of how it can be potentially harmful to humans. Condition Transformation example a. Beneficial b. Harmful 9. Provide a rational or benefit of adding DNA sequences coding for fluorescent proteins such as GFP, to tag genes of interest in plasmids used for transformation. 10. In your own words, explain the process of transformation. 9

11 BABEC Educational Transformation Kits For Research Use Only. Not for use in diagnostic procedures. BABEC thanks Qiagen for their generous support of plasmid prep kits for BABEC bacterial transformation labs. 10

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