Introduction to Genetic Engineering Bacterial Transformation with Green Fluorescent Protein (pglo)

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1 Introduction to Genetic Engineering Bacterial Transformation with Green Fluorescent Protein (pglo) Table of Contents Bacterial Transformation Lab Activity Introduction Background Information and Scientific Theory. 2 General Lab Skills Required 4 Laboratory Activity Worksheet: Bacterial Transformation Worksheet: Calculating Transformation Efficiency Appendix: Streaking starter plates for E.coli.14 Acknowledgements..15

2 Introduction to Genetic Engineering Bacterial Transformation with Green Fluorescent Protein (pglo) Genetic engineering is an umbrella term that encompasses many different techniques for moving DNA between different organisms. Transformation is the process by which an organism acquires and expresses a whole new gene. In this activity, you will have the opportunity to genetically transform bacteria cells; altering them so that they can make an entirely new protein. This procedure is used widely in biotechnology laboratories all over the world, enabling scientists to manipulate and study genes and proteins in exciting new ways. Adding a new gene to bacteria cells has become a relatively simple process. You will add a gene that codes for Green Fluorescent Protein (GFP). This protein was discovered in the bioluminescent jellyfish called Aequorea victoria, a jellyfish that fluoresces and glows in the dark (Figure 1). The gene for GFP was isolated in 1994 and was quickly used in laboratories as a way to brightly label proteins in a living cell. This tagging of proteins allowed researchers to visualize the location of specific proteins to learn more about their biological functions in exciting new ways. The discovery of GFP proved to be so important that the Nobel Prize in Chemistry in was awarded to Osamu Shimomura, Marty Chalfie and Roger Tsien in 2008 for their work. Since then, Roger Tsien s laboratory at UCSD has altered the GFP gene to make a full rainbow of proteins. Figure 2 shows how bacterial expressing many different colored fluorescent proteins can be grown together on one plate. Figure 1 Figure 2 Aequorea victoria A rainbow of fluorescent glowing under UV light growing on an agar plate Bacteria are commonly used for genetic transformation experiments because they are simple, single-celled organisms that grow and reproduce very quickly. Bacteria cells store their DNA on one large, circular chromosome. But they may also contain one or more small circular pieces of DNA called plasmids. Plasmids are able to replicate independently of the large bacterial chromosome, and can transfer easily between cells. Figure 3 shows the circular DNA chromosome and plasmid DNA inside of a cell. Figure 3 Genetic material in bacteria takes 2 forms Bacterial evolution and adaptation in the wild often occur via plasmid transfers from one bacterium to another. An example of bacterial adaptation is resistance to antibiotics via the transmission of plasmids. 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 or damage from insects, and to increase their absorption of nutrients. Toxic chemical spills can often be bioremediated (cleaned-up) by transformed bacteria specifically engineered to do the task. Currently, many people with diabetes rely on insulin made from bacteria transformed with the human insulin gene. Scientists use transformation as a tool to study and manipulate genes all the time. 1

3 Background Information and Scientific Theory The Central Dogma of Molecular Biology A basic tenet of biology, from single-celled bacteria to eukaryotes, is the mechanism of coding, reading and expressing genes. The central dogma of molecular biology states that: DNA > RNA > PROTEIN > TRAIT. This curriculum is an example of the central dogma in action. The instructions for GFP production are encoded in the DNA. When transcription is turned on, the cell turns those instructions into an mrna transcript. This transcript is then translated into protein, which provides the trait of fluorescence. Gene Regulation Every cell in the human body shares an identical genome that contains over 20,000 different genes. But if all cells have the same genes, how is it that a muscle cell ends up being so very different from a brain cell? The answer lies in the fact that there is a specific process for controlling which genes are turned on and which are turned off in every single cell. Gene regulation is the name for all the different cellular processes that have to take place in order for a gene to result in a protein. Gene regulation is an important concept in biology dictating where and when genes are turned on or off. Gene expression occurs when genes are turned on, resulting in the expression of proteins the workhorses of the cell. Proteins called transcription factors are frequently used by cells to turn transcription on or off depending on environmental conditions. They are important for cellular development, tissue specialization, and organismal adaptation to the environment. Transcription factors act at the promoter region in front of a gene. At the promoter, RNA polymerase initiates transcription and turns a gene on; the gene is then said to be expressed. Once the mrna transcript is made, it can be translated into protein (see Fig 5). All the genes in our bodies are highly regulated to allow for maximum efficiency, and to decrease waste (energy) in our cells. The pglo System In this laboratory activity, you will have the opportunity to genetically engineer a cell and you will see with your own eyes the critical role of gene regulation in living systems. This is because the expression of the GFP gene in this experiment is not automatic. Rather, it happens only when the environmental conditions are just right. Plasmids used by molecular biologists are named with an acronym that begins with the lower case "p", and followed by a name that conveys information about its function. pglo is the name for a plasmid that has been engineered to contain the gene for GFP, which glows under UV light. Using recombinant DNA technology, scientists designed this plasmid to contain two additional genes, for a total of three genes whose function it is important to understand before beginning this activity. Figure 4: The pglo plasmid has been engineered to express 3 genes amp r ara ara Codes for the regulatory protein arac, which works with the sugar arabinose to turn on GFP transcription by recruiting RNA polymerase GFP Codes for Green fluorescent protein, which is derived from Aequorea victoria - a bioluminescent jellyfish that fluoresces under UV light. amp r Codes for the enzyme beta-lactamase, which inactivates the ampicillin and allows the cell to grow in the presence of that antibiotic. 2

4 In this lab activity, you will be inserting pglo into non-pathogenic E. coli bacteria. The procedure is never 100% efficient and only a few of your E. coli bacteria will successfully take up the pglo. How will you know which cells contain the plasmid? pglo contains a gene that codes for a protein that protects the cell against the toxic effects of antibiotics. This means that only cells that have the plasmid will survive in the presence of antibiotics. In this procedure, we use ampicillin, an antibiotic very similar to penicillin. This step is called antibiotic selection, and it allows you to select only the cells that have been transformed. The beta-lactamase gene in pglo codes for a protein that breaks down ampicillin. Expression of the beta-lactamase gene in cells that have been successfully transformed allows them to grow in the presence of ampicillin. Non-transformed cells will die or not grow into visible colonies. Your transformed cells will grow on a plate with ampicillin, but they will not fluoresce green until the GFP gene is turned on. Here s where the idea of gene regulation comes into play. Transformed cells will grow on plates not containing arabinose, but will only fluoresce green under UV light when arabinose is included in the nutrient agar. Therefore, arabinose, a sugar that bacteria consume for energy, is the critical ingredient for making your bacteria glow. What s so special about arabinose? It teams up with the arac, the regulatory protein that is expressed by pglo. Regulatory proteins control the timing and location of many cellular processes. Specifically, arac is a transcription factor which, as described previously, functions to turn genes on and off. But it can t turn GFP on by itself it needs the help of arabinose. Together, they work to bring in RNA polymerase, the enzyme that makes RNA, and only then can the glowing, green protein be made. It's a finely orchestrated dance, and all the right players have to be in place for success. Figure 5 Gene regulation of GFP in pglo Figure 5 shows that when arac teams up with arabinose, its shape changes. The protein arac easily forms a bond with the sugar arabinose, and only when they both get close together can the complex function as a transcription factor. What it then does is very simple: it stimulates RNA polymerase to start transcription, and we see firsthand the central dogma of molecular biology in action! The Transformation Procedure In order to increase the chances that your E. coli will incorporate foreign DNA, you will need to alter their cell membranes to make them more permeable. This is a two-step process. First you place your cells and pglo together in a transformation solution (which contains calcium chloride) to neutralize the charge. Second, you quickly heat shock them with a temperature change (42 o C). This hot temperature permeabilizes (loosens) the bacterial cell wall, making it easier for pglo to cross it. This process can be harmful to the cells, so you want to give them a nutritious broth to restart their growth as soon as you re done. Luria Broth (LB) is a liquid that contains proteins, carbohydrates and vitamins so that the E.Coli can rapidly recover and thrive. They will then be placed on an agar medium, a jello-like substance containing LB, with or without antibiotic or sugar, to grow overnight. 3

5 General Lab Skills Required for Success Using Sterile Technique Students should wash their hands before starting lab, after handling recombinant DNA organisms/containers, and before leaving the lab area. All lab surfaces should be decontaminated at least once a day during each class section and following spills. Students should avoid touching the tips of the pipettes or inoculating loops onto any contaminating surfaces, unless instructed in the protocols. Students should practice proper aseptic techniques to prevent contamination. Using Microipettes Students need to be familiar with micropipetting techniques and remember to exchange pipet tips to avoid cross contamination. Do not carry micropipettes sideways or upside down while transferring liquids. Please don t abuse the micropipettors by dialing in amounts beyond their intended calibration limits. When transferring liquids, the student holding the micropipettor should also be holding the microfuge tube of liquid to transfer. Both should be brought to eye level in order to visually confirm that liquid has been transferred. A teammate should confirm the correct micropipettor setting, correct tube of liquid to transfer and the use of clean pipet tips. Success of the lab depends on the proper use of tools and reagents required for the protocol. UV Safety Ultraviolet radiation can cause damage to eyes and skin. Use UV-rated safety glasses or goggles if looking directly at UV light. Using Experimental Controls In this lab, it is important to confirm which cells have received the plasmid, and under which conditions the green fluorescent proteins are being produced. You will need to prepare a series of experimental controls to be able to interpret your results correctly. These controls are designed to minimize the effects of factors other than the single concept that you are testing. Therefore, 2 different reactions will be performed: one with pglo plasmid (+pglo) and one without it (- pglo). See Figure 6 to understand how to set up your reactions. Figure 6: Bacterial growth conditions Nutrient Agar (LB) Antibiotics (ampicillin) On Switch (arabinose) E.Coli + pglo E.Coli - pglo #1 #2 #3 Yes Yes Yes Yes No Yes No The - pglo control serves two roles: 1) to ensure that the bacteria are still alive after the chemical and heat shock procedure, and 2) to make sure that the ampicillin is working property. You will plate these bacteria under conditions #1 and #2, but you should only expect them to grow in condition #1. The +pglo transformation will grow in condition #1 and #2. However, only the bacteria that successfully took up the pglo plasmid will grow in condition #2. In this reaction, you will observe the process of antibiotic selection, but you should not see any GFP produced. Condition #3 is only used for the +pglo reaction. This example proves that the transcriptional control of the GFP gene is intact. The bacteria on this plate are the only ones that should glow green when exposed to UV light. In this reaction, you will observe the process of gene regulation. 4

6 The protocol outlined next describes the procedure for adding plasmid DNA to a bacterial cell. Be sure to follow each step very carefully. You will be cooling your E. Coli cells on ice, then heating them in a water bath, then letting them recover. Make sure your pipetting volumes are accurate at every step. Afterwards, you will grow the cells on a petri dish containing LB agar, antibiotics and arabinose. After 1-2 days, you will look for the development of green fluorescent colonies of bacteria. Student Learning Outcomes at the end of this laboratory, students will be able to: 1. Describe the central dogma of molecular biology. 2. Explain the process of bacterial transformation and selection. 3. Relate the use of bacterial transformation in biotechnology. 4. Differentiate transformed from non-transformed cells. 5. Calculate transformation efficiency and compare with the class data. Preliminary predictions and questions to think about Will the untransformed bacteria appear neon green under a UV lamp? Why or why not? Why don t you attempt to grow the pglo reaction under LB/ara? Do you expect the same number of colonies for the +pglo reaction under condition #1 than condition #2 (on page 4?) Before beginning the transformation, observe a plate of E. coli and a vial of pglo plasmid under a UV lamp. Then view your transformed colonies once you complete the lab activity. Do you see glowing? Fill out the table below: Item Prediction View with UV Lamp Explanation of Results E. coli growing in petri dish on LB agar Vial of pglo plasmid Transformed E. coli grown under condition #2 5

7 Laboratory Activity Place a check mark in the box as you complete each step. 1. Sterilize lab surfaces and wash hands before beginning the lab. 2. Obtain one empty 1.5mL microfuge tubes from your instructor. Using a permanent marker, label the tube +DNA and team intials. **Negative Control: Assigned groups will perform a mock transformation to be used as negative control for the class. Label a second tube DNA. Label each tube twice, on the lid and on the side. Place these tubes into a Styrofoam cup containing crushed ice. pglo Transformation Protocol 3. Add 250µL of Transformation Solution (TS) to each tube using a proper micropipette (Alternatively you can use a plastic transfer pipette) Note: TS contains calcium chloride (CaCl2), 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. Place the plate on the UV lamp and observe the colonies. Are they glowing? 250µL TS UV Light Wear safety glasses while using the UV lamp. 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 or rack the tube. Make sure there are no lumps. If doing the negative control, 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 pglo under a UV lamp. Does it glow? UV Light 6

8 7. With a P-20 micropipettor, transfer 10µL of the pglo plasmid into your tube labeled +DNA only. (Alternatively, use the 10µL inoculation loop. Dip the loop into the 1mL stock plasmid tube. A noticeable film will form around the ring due to surface tension (like a bubble wand). Swirl the loop into tube labelled +DNA.) **DO NOT add plasmid to the DNA tube. Close the cap and flick the tube to mix. 10µL pglo + DNA 8. Incubate both tubes on ice for 10 minutes, making sure the tubes are in contact with the ice. + DNA DNA Incubate 10 minutes on ice 9. While you re waiting, pick up these 3 plates: 1 LB, 1 LB/amp, 1 LB/amp/ara On the outer edge on bottom (non-lid side) of the plate, write +DNA. **If performing the negative control experiment, pick up 1 LB and 1LB/Amp plate. Label them DNA. Also write your team initial or symbol and the date on the bottom (non-lid side) of each plate. PGlo Transformation +DNA LB LB/amp LB/amp/ara *Negative control -DNA LB LB/Amp **only for groups assigned to do the negative control 10. Taking your tubes on ice to the water bath, 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. Make sure the tubes are pushed down as far as they can go in the rack to contact the hot water. 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. + DNA DNA + DNA DNA Water Bath 42 C / 50 seconds + DNA DNA 2 min 11. Return your tubes to a tube rack now resting on your lab bench. Using a proper micropipette (or transfer pipette), add 250µL of LB broth to each of the tubes 250µL LB Remember to change the tips between the tubes! 7

9 12. Close the tubes. Mix each tube by flicking it several times with your finger. Incubate the tubes for at least minutes at 37 C. You can use the bacterial incubator or other warm place like the top of a refrigerator or keep the tube warm in your hands for this step. This process will allow the transformed bacteria to recover by providing nutrients for their growth. Incubate minutes at 37 C 13. Obtain your labeled plates. Using a P200 (or sterile transfer pipette), transfer 150µL of the +DNA directly to the agar into each plate labeled +DNA plate. Be careful not to poke into the agar! 14. Using a clean inoculation loop, gently spread the liquid on the agar of each plate. You may use the same loop for all the +DNA plates. Be careful not to poke into the agar! Evenly cover as much of the plate as possible. PGlo Transformation +DNA LB LB/amp LB/amp/ara *only for groups doing the neg controls Discard used loops into a waste container with disinfectant. Allow bacteria to soak into the agar plate for a few minutes before the next step. 15. If performing the negative control experiment, repeat step 13 and 14 using the -DNA on the appropriately labeled -DNA plates. Use a clean transfer pipet and inoculation loop for this set. Negative control -DNA LB LB/Amp 16. Invert your plates (lid on bottom). Then stack and tape them together. Make sure to use 2 pieces of tape, one on each side of the stack. Place plates into an incubator oven set at 37 C until the next day or when colonies are visible. Alternatively, stack the plates in a warm spot in the classroom. It may take 2-3 days for bacterial colonies to appear. After the colonies have appeared, you may keep the plates by wrapping them in parafilm and storing in the refrigerator. 17. Clean lab station. Decontaminate all lab surfaces with dilute disinfectant and wash hands following the lab! 8

10 Name Date Period Worksheet: Bacterial Transformation Lab Predictions Will the untransformed bacteria, pglo 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 pglo plasmid Transformed E. coli colony 2. Explain the purpose of these processes or substances during transformation. Process or Purpose Substance a. LB agar b. Ampicillin or antibiotic c. Calcium chloride d. Heat shock e. Arabinose 3. Describe 2 differences and 2 similarities between these Bacteria. Condition - pglo DNA bacteria + pglo DNA bacteria Difference Similarity 9

11 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, +DNA LB/amp, +DNA LB/amp/ara, +DNA Predictions* Illustration of Results Description of Results 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. 6. Explain what may have occurred to produce these results. ( = colony) Contents LB -DNA LB/amp -DNA LB/amp/ara +DNA Illustration of Results Description of Results Possible explanation for results 10

12 Worksheet: Calculating Transformation Efficiency When performing transformation experiments, you usually want to obtain as many transformants as possible. This is important because you want to make sure your conditions for transformation is at its optimum. Transformation efficiency is the efficiency whereby cells take up the introduced DNA. Many factors contribute to transformation efficiency: cell age and competency (the ability to take up DNA), the type of cells being transformed, plasmid length and quality, the method of transformation (heat shock or electroporation) and just different conditions in general. Having a low transformation efficiency may point to poorly competent cells, poor conditions, or poor techniques (not following protocol). In a research lab, it s good to have many transformants for research, just in case individual transformants may not work as well (e.g. different levels of expression), or some other unknown problems associated with transformed cells. In making a genomic library, you want as many transformants as possible to have a robust library. In cell culture, you may take a population of transformed cells for further study therefore having a high transformation efficiency allows for better study. In this exercise, we will calculate the transformation efficiency of the E. coli bacteria by pglo. The data can then be gathered from each team of the class and the data compared with a different transformation technique called electroporation. Transformation efficiency calculation: The number of colonies observed growing on an agar plate (cfu) Amount of DNA used (in µg) cfu=colony forming units Two data are needed for this: 1. Total number of green fluorescent colonies on your LB/amp/ara plate. 2. Total amount of pglo plasmid DNA used for bacterial transformation that was spread on the LB/amp/ara plate. 1. Determine the total number of transformed green fluorescent colonies. Place the LB/amp/ara plate near a UV light source. Count the number of green fluorescent colonies that glow under UV light. Enter that number here Total number of colonies = 2. Determine the amount of pglo DNA in the cells spread on the LB/AMP/Ara plate. Two pieces of information are needed: a) The total amount of DNA you used for the +DNA in the experiment b) The fraction of DNA that was spread onto the LB/amp/ara plate a. Total amount of DNA: DNA in µg = (concentration of DNA in µg/µl) x (volume of DNA in µl) In this experiment, 10µl of pglo at a concentration of 0.01 µg /µl was used. Enter that number here Total amount of pglo DNA, µg used in this experiment = 11

13 b. Fraction of pglo plasmid DNA (in the bacteria). For this experiment, a certain amount was spread onto each plate. To find that fraction: Fraction of DNA used Sample volume spread on LB/amp/ara plate, in µl Total sample volume in tube, in µl 150µl of cells was spread from the tube containing a total volume of 500µl of solution. Enter that number here Fraction of DNA= c. How many µg of pglo DNA was spread on the LB/amp/ara plate? Multiply the total amount of pglo DNA used by the fraction of pglo DNA you spread on the LB/amp/ara plate. pglo DNA spread (µg) = amount of DNA used (µg) x fraction of DNA Enter that number here pglo DNA spread, µg = Now, we are finally ready to calculate the transformation efficiency! Number of colonies on LB/amp/ara plate = pglo DNA spread, µg = Transformation efficiency calculation: The number of colonies observed growing on an agar plate Amount of DNA used (in µg) Enter that number here Transformation efficiency = transformants or cfu/µg cfu=colony forming units 12

14 Analysis of results: What is the transformation efficiency of each team in the class? Team Efficiency Do you see differences between the teams? Why do you think there are these differences? What could increase/decrease transformation efficiency? In past studies, this method of heat shock protocol that was performed by research labs usually has a transformation efficiency between 8x10 2 and 7x10 3 transformants per microgram of DNA. How does your team s result compare to this data? How does the class result compare to your data and to the data by research labs? Another method for transformation is called electroporation. In this method, an electric field is applied to allow the cell membrane to open up and take up DNA. The transformation efficiency from electroporation may be 1x10 8 cfu/μg. What fold higher is the transformation efficiency by electroporation vs. heat shock? 13

15 Appendix: Streaking starter plates of E. coli Starter plates are needed to produce bacterial colonies of E. coli on agar plates. LB agar plates should be streaked to produce single colonies and incubated at 37 C for hours before the transformation investigation begins. Under favorable conditions, one cell multiplies to become millions of genetically identical cells in just 24 hours. There will be millions of individual bacteria in a single millimeter of a bacterial colony. Depending on time, you may prefer your students to learn how to streak their own plates for individual colonies. Plate Streaking Streaking takes place sequentially in four sections. The first streak spreads out the cells. In subsequent streaks the cells become more and more dilute, thus increasing the likelihood of producing single colonies. 1. Draw quadrants on the underside of the petri dish. Using a sterile inoculation loop or sterile pipet tip, pick up one bacterial colony from live E. coli culture plate. 2. Using a back and forth motion, gently spread the colony into one quadrant of the LB starter plate. Keep the lid slightly tilted open - only as much as necessary. Be careful not to puncture the agar. 3. Rotate the plate one-quarter of a turn. Go into the previous streak about two times and then back and forth as shown for a total of about 5-10 times. 4. Again, rotate the plate one-quarter of a turn and pass over a previous streak from the previous quadrant several times with the loop. 5. Repeat step 3, but this time, drag out the loop to form a tail not touching any previous streaks. Close your plate to avoid further contamination. 6. Place the used loop (or tip) in a disinfectant solution waste cup. Follow this procedure for the remaining starter plates. Once starter plates are inoculated, incubate them upside down in a 37 C incubator oven for 24 to 36 hrs. 7. If your students are not using the plates right away, seal the sides with Parafilm or lab tape so they don t dry out, invert the plates, and place them in a dark cupboard until needed. Avoid refrigerating your starter plates as cooling will reduce your transformation efficiency. What to expect the next day You should see individual bacterial colonies in quadrant 4, and very dense bacterial growth in quadrant 1. Quadrants 2 and 3 will have bacterial density somewhere in between, similar to what is seen below: Your streaked plate should look similar to this image after hours. Note: the images on this page have been provided by the Florida Institute of Technology 14

16 BABEC Educational Transformation Kits BABEC thanks Qiagen for their generous support of plasmid prep kits for BABEC bacterial transformation labs. Acknowledgements The following images have been provided courtesy of: Figure 1 adapted from National Geographic. Figure 2 Tsien Laboratory at UCSD. Figure 3 Wikipedia. Figure 4 adapted from Bio-Rad. 15