Transformation and Protein Purification of GFP Kathryn Bizier, Malden High School 2014 Summer Research Internship Program Hynes Lab, Koch Institute Introduction: This summer I helped with a project that uses zebrafish as a model for studying human melanoma metastasis. Specifically, I helped with the portion of the study that involved cloning desired genes that could then be inserted into the fish cells and then visualized in various ways. One of the genes that are used to visualize fish cells is the Green Fluorescent Protein, or GFP. Since actual animal work is not possible within the high school setting, I thought that having students transform bacteria with the GFP gene, and then purifying the gene would introduce students to some of the ideas and protocols that I used over the course of my 6 weeks. While protocols exist (and are referenced within the lesson plan), the goal was to develop useful power points, worksheets, and lab handouts to assist students in transforming cells with the GFP gene, and then purifying the protein. I also hoped to familiarize students with plasmid structure, as well as the New England Bio Labs website. In the following pages, you will find protocols that have been put together to accomplish this task, as well as a plasmid worksheet, PowerPoint presentations, and “protocol cards” which students can use to help them visualize the different steps in the transformation process. I have the lesson plan first, with the two lab protocols following. At the end of the document you fill find the plasmid worksheet. PowerPoint presentations are also listed on the website. Kathryn Bizier, Malden High School th
Grade Level 10-­‐12 Unit: Genetic Engineering/Recombination Techniques Objectives: 1. SWBAT use manipulatives to plan out the transformation protocol 2. SWBAT use proper lab techniques to transform bacteria with the GFP gene 3. SWBAT use chromatography protocol to isolate and purify the GFP gene 4. SWBAT identify uses of transformation in the biotechnology world Timing: 1st Day: Transformation PowerPoint; Manipulative Exercise & Pour agar plates for day 2 (45 – 90 minutes, depending on whether you have students pour the plates and how comfortable doing so) 2nd Day: Transformation Protocol & Plate bacteria; chromatography PowerPoint (90 minutes) 3rd Day: Examine plates, Select Colonies (Can do chromatography PowerPoint here as well 4th Day: Protein Chromatography & Analyze Results (45 minutes) NGSS/MA Standards: HS-­‐LS1-­‐1. Explain that genes are regions in the DNA that code for proteins, which carry out the essential functions of life. Construct a model of transcription and translation to explain the roles of DNA and RNA in coding the instructions for polypeptides, which make up proteins. Explain that different classes of proteins regulate and carry out the essential functions of life. HS-­‐LS1-­‐8(MA). Explain how the structure of DNA, including its spiral shape and paired nucleotides, is related to its function of storing and transmitting hereditary information. HS-­‐LS1-­‐9(MA). Research and communicate information about features of virus and bacteria reproduction and adaptation to explain their ability to survive in a wide variety of environments. HS-­‐PS1-­‐11(MA). Construct an argument to show differences in the atomic composition and molecular geometry of substances that allow for identification, detection, and separation of substances in a mixture. NGSS Science & Engineering Practices: 1. Practice 1 – Asking Questions and Defining Problems 2. Practice 4 – Analyzing and Interpreting Data Background knowledge • Students should know that genes can be found on chromosomes, and that they generally code for proteins • Students show know the basic structure of a prokaryotic cell, and how it compares to the structure of a eukaryotic cell • Students should know what recombinant DNA is • Students should know the basics of the central dogma – how genes get expressed as proteins • Skill: Students should know how to use a micropipette properly, as well as how to plate bacteria Kathryn Bizier, Malden High School Day 1 Objective: SWBAT to outline the basic steps of transformation, and explain the purpose of a plasmid. Possible Resources to use: • Bozeman Biology podcast on Lab 6 AP https://www.youtube.com/watch?v=OZyFX9megs8 • Youtube video on plasmids: https://www.youtube.com/watch?v=GNMJBMtKKWU 5 minutes: Do-­‐Now/Warm Up Ask students to answer the following questions and then have a class discussion 1. What type of organisms is E. coli? 2. What cellular structures can be found in an E. coli cell? 3. Why might you want to make an organism take up additional (“extra”) DNA? 15 minutes: PowerPoint on Transformation • Have students take notes on the slides as this will help them with the lab • Time saving technique: provide students with guided notes to fill in 25 minutes: Have students pour agar plates (if you choose to do so) • Make sure students wear safety goggles, and use rubber grips to hold molten agar • Explain how you will store the plates once they are cooled • Time-­‐saving suggestions: mass out agarose ahead of time, label plates ahead of time 20-­‐30 minutes: Students use card manipulatives to work through the transformation protocol • Have students try to figure out the order before giving them the actual lab instructions • If time allows, have students create a flow chart that shows the protocol that they will be following Homework: Have students do the background reading for the protocol and mark-­‐up the text. Then have them answer the pre-­‐lab questions. This can be used as a requirement in order for the students to be able to do the lab the next day. Day 2 Objective: SWBAT transform bacteria with the GFP protein using the lab protocol provided to them 5 minutes: Do-­‐Now/Warm Up Have students pair-­‐share their answers to the pre-­‐lab questions. Address any concerns/questions that students have before commencing with protocol. Also, use this time to prep the students on any safety concerns and/or parts of the protocol that need to be addressed 40 minutes: Lab Protocol day 1 Kathryn Bizier, Malden High School Note: If you are using kanaymycin, you will need to allow for extra class time since there needs to be recovery time. 10 minutes: Clean up 15 minutes: Chromatography PowerPoint • Note: If you have a shorter class period, you can save the chromatography PowerPoint for tomorrow Homework: Have students answer the pre-­‐lab questions for “day 2” of the transformation protocol. Also have them complete the pGLO worksheet. Day 3 All students need to do on this day is examine the plates, and select colonies, which should take 15-­‐20 minutes, so you can do this in addition to another class plan. Or, as stated previously, you can also do the chromatography PowerPoint here as well, and take up close to a whole 45 minute class period. Homework: Have students read the introductory reading to the chromatography lab & have them mark-­‐up the text. If they did not finish the analysis questions in class, have them finish those tonight as well.sd Day 4 Objective: SWBAT use the protein chromatography protocol to purify the GFP protein. 5 minutes: Do-­‐Now/Warm UP Ask students the following questions 1. What do the terms hydrophobic & hydrophilic mean? 2. What does it mean to purify something? 3. Why might you want to be able to purify a protein from a mixture? 25-­‐35 minutes: Chromatography Protocol 15-­‐20 minutes: Clean up & Analysis of Data Homework: Have students finish the analysis questions if they were not finished in class. Assessments: Questions within the lab protocols, as well as the analysis questions at the end of the labs. Kathryn Bizier, Malden High School Kathryn Bizier, Malden High School Bacterial Transformation with GFP Teacher Background Information Depending on time limits and what you’d like students to experience, you can either pour the plates ahead of time, or have students pour the plates. Pouring Plates (Adapted from Addgene’s Protocol – will make about 25 total plates) Protocol To make 500mL of LB agar (makes about 25 LB agar plates): 1. Weigh out the following into a 1L Erlenmeyer flask: 5g NaCl 5g Tryptone 2.5g Yeast Extract 7.5g Agar and (dH2O) to 500mL 2. Swirl to mix -­‐ the contents do not have to be completely in solution, but any powder left on the sides of the flask will caramelize on the glass during autoclaving. •
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3. Place the flask into the microwave, and microwave for 3-­‐5 minutes, keeping a close eye on the agar, you don’t want it to spill over! 4. Check the flask to make sure all bits are dissolved, and remove it once it is totally molten. 5. After removing the solution from the microwave, allow the agar solution to cool to 55°C. Note: This can be done by placing the flask in a 55°C oven or water bath, as this will hold the temperature and it can be left unattended for some time. 6. While the medium is cooling, label ½ of the plates +amp, and label the other half –amp 7. Using a fresh 10mL pipet, pour 5mL of the agar into the –amp plates Note: Pour slowly from the flask into the center of the petri dish. When the agar has spread to cover about 2/3 of the dish stop pouring and the agar should spread to cover the entire plate. You may need to tilt the plate slightly to get the agar to spread out completely. If you pour in too much, the plate will be fine, but it will reduce the number of plates you can make per batch. Note: If bubbles are introduced during the pouring, these can be removed by quickly passing the flame of an inverted bunsen burner over the surface of the plate. Be careful, if you leave the flame too long it will melt the petri dish. Also be careful not to burn yourself. 8. Add the ampicillin to the solution (500μL if you are using a 1,000x antibiotic stock) and swirl to mix. Kathryn Bizier, Malden High School Note: 1000x stock is made by adding 500 mg ampicillin powerd to 10 mL 50% Ethanol/dH2O (5mL 100% ETOH, 5 mL dH2O). 9. Using a fresh 10mL pipet, pout 5mL of the agar + ampicillin to the remaining +amp plates. 10. COVER and WAIT at least twenty minutes for the LB-­‐agar plates to solidify. For optimal results, leave plates at room temperature overnight. 11. STORE plates at room temperature for no more than two days. Plates should be inverted and placed in a sealable plastic bag to ensure that they do not dry out. NOTE: If plates are prepared more than two days before use, they should be stored inverted in a plastic bag in the refrigerator (4°C). Remove the plates from the refrigerator and warm in a 37°C incubator for 30 minutes before use. Safety Concerns: You should research if any of the students in your class have allergies to penicillin, ampicillin, kanamycin or tetracycline. If so, you should speak to the school nurse and the parents about students participating in the lab. Students should wear gloves and goggles at all times during this lab. Material notes: Make sure that the plasmid you obtain has both the GFP and ampicillin resistance gene on it. Bio-­‐Rad can supply this in the form of the p-­‐Glo plasmid. One option would be to have the students insert the GFP gene into a plasmid as well. This would involve researching which restriction enzymes to use, which plasmid to use, etc. Kathryn Bizier, Malden High School Part 1: Transformation of Bacteria Introductory Reading (Adapted from: http://web.pdx.edu/~yanm/pGLOlab.pdf) Green Fluorescent Protein (GFP) is a protein produced by the bioluminescent jellyfish Aequorea victoria. Green Fluorescent Protein causes the jellyfish to fluoresce and glow in the dark. Remember that a gene is a piece of DNA that provides the instructions for making (codes for) a protein. The protein encoded by the gene gives an organism a particular trait. In this lab you will start with a procedure known as genetic transformation. Genetic transformation literally means change caused by genes, and involves the insertion of a gene into an organism in order to change the organism’s trait. Genetic transformation is used in many areas of biotechnology. In agriculture, genes coding for traits such as frost, pest, or spoilage resistance can be genetically transformed into plants. In bioremediation, bacteria can be genetically transformed with genes enabling them to digest oil spills. In medicine, diseases caused by defective genes are beginning to be treated by gene therapy; that is, by genetically transforming a sick person’s cells with healthy copies of the defective gene that causes the disease. In the first week you will use a procedure to genetically transform bacteria with the gene that codes for Green Fluorescent Protein. Following the transformation procedure, the bacteria express their newly acquired jellyfish gene and produce the fluorescent protein, which causes them to glow a brilliant green color under ultraviolet light. During the next lab section, after growing the bacteria up, you will lyse the bacteria open, and then purify out the GFP protein using a column. Fundamental to the process of genetic transformation of bacteria are plasmids. In addition to one large chromosome, bacteria naturally contain one or more small circular pieces of DNA called plasmids. Plasmid DNA usually contains genes for one or more traits that may be beneficial to bacterial survival. In nature, bacteria can transfer plasmids back and forth allowing them to share these beneficial genes. This natural mechanism allows bacteria to adapt to new environments. The recent occurrence of bacterial resistance to antibiotics is due in large part to the transmission of plasmids. The plasmid (Fig. 1, not a naturally occurring plasmid) contains the gene for GFP, and also contains the gene for the enzyme b-­‐lactamase (bla), which provides resistance to the antibiotic ampicillin. The b-­‐lactamase protein is produced and secreted by bacteria that contain the plasmid. b-­‐lactamase inactivates the ampicillin present in the LB nutrient agar, allowing bacterial growth. Only transformed bacteria that contain the plasmid and express b-­‐lactamase can survive on plates that contain ampicillin. Only a very small percentage of the cells take up the plasmid DNA and are transformed. Untransformed cells cannot grow on the ampicillin selection plates. The transformation procedure involves three main steps. These steps are intended to introduce the plasmid DNA into the E. coli cells and provide an environment for the cells to express their newly acquired genes. 1) Cells are first treated with a solution of CaCl2, which is thought to neutralize the repulsive negative charges of the phosphate backbone of DNA and the phospholipids of the cell membrane, allowing the DNA to adhere to the cells. This is referred to making the cells ‘competent’ (i.e. the cells are capable, or competent, to take-­‐in exogenous DNA). 2) Cells are then subjected to a heat shock, which is thought to increase the permeability of the cell membrane, allowing the cells to take-­‐in the plasmid DNA. 3) Cells are allowed to grow in a ‘recovery’ phase in the absence of ampicillin. During this time the cells begin to produce the b-­‐lactamase protein, which will allow them to survive when they are subsequently placed on agar plates containing ampicillin. Kathryn Bizier, Malden High School Important Safety Information! 1. IF you are allergic to penicillin, ampicillin, kanamycin or tetracycline, talk to your teacher. 2. Gloves and goggles should be worn. 3. Exercise extreme caution when working with equipment which is used in conjunction with the heating and/or melting of reagents. 4. The E. coli bacteria used in this experiment is not considered pathogenic. Although it is rarely associated with any illness in healthy individuals, it is good practice to follow simple safety guidelines in handling and disposal of materials contaminated with bacteria. 5. Properly dispose of materials after completing the experiment. -­‐ SEE YOUR TEACHER FOR THIS!!! 6. Wash your hands after taking off your gloves! Pre-­‐Lab Questions 1. Why might we want to insert DNA into bacteria? 2. Can you think of ways we might be able to tell if bacteria have taken DNA up? 3. What is a plasmid? 4. What is ampicillin? 5. Why might it be useful to give an organism the ability to glow (besides just the fact that it looks cool?) 6. How are bacteria cells different from eukaryotic cells like human cells? Kathryn Bizier, Malden High School Materials (per group): Tube of plasmid DNA 1 Tube of LB broth Two tubes of E. coli bacteria (on ice) Micropipette & Tips 1 LB agar plate 1 LB agar plate w/ selection (+amp) Inoculation loops Transfer pipette Option: Have students pour their own plates if you’d like them to experience preparing agar plates (see protocol in teacher information) Materials for entire class: Water bath at 42°C Ice Biohazard Waste Bag UV Lights 37°C incubator Protocol (Adapted from Edvotek’s 221 Protocol & web.pdx.edu/~yanm/pGLOlab): 1. Label one tube of your E. coli bacteria +GFP and the other –GFP, along with your groups’ initials. Keep these on ice when not in use! 2. Examine your tube of GFP plasmid DNA with the UV lamp. What do you see?!! Make a note of your observations here: 3. Pipette 20 μL of GFP plasmid DNA into the E. coli tube +GFP. Mix by gently flicking on the side of the tube with your finger. Return the tube to ice! Question: Why did you not add plasmid DNA to the –GFP tube? 4. Put the tubes on ice for 10 minutes. Question: Why do you think we put the tubes on ice? 5. While the tube is on ice, your group should obtain one LB agar plate and one LB agar + ampicillin (+amp, the one with a stripe). Write your group’s initials on the plate. Make sure you label on the BOTTOM of the plates! Question: Why do we have the two different plates? I.e. what purpose does each plate serve? Kathryn Bizier, Malden High School 6. Take the tubes off the ice, and put them directly into the 42°C water bath for 50 seconds, using one of the blue floating racks to hold your samples. Make sure your push your tubes all of the way through the rack so the bottom of the stubs stick out and make contact with the warm water. Question: What purpose does heating the tubes serve? 7. Take the tubes out of the water bath, and put the tubes directly onto ice for 2 minutes. Make this transfer as quick as you possibly can! 8. Remove the tubes from the ice and put them in a holder on the bench top. Open each tube, and using a new sterile pipet tip each time, add 250 μL of LB nutrient broth to the tube and reclose it. Note: If using kanamycin, the cells should be allowed to recover at 40 minutes. If using ampicillin, you can proceed directly to step #9. Question: What purpose does the LB broth serve? Question: What do you want the cells to be at room temperature? 9. Tap the closed tubes with your finger to resuspend the cells evenly throughout the solution. 10. Using a pipet, transfer 10 μL of the +GFP onto your LB agar + ampicillin plate and then transfer 10 μL of the – GFP suspensions onto your LB agar plate. 11. Spread the solutions on the plates using the sterile inoculation loops, by gently skating the loop along the surface – use a new sterile one for each plate! Be careful not to stab the agar. Question: Why do you need to use separate inoculation loops for the plates? 12. Stack up your plates and tape them together. Put your group’s names and class period on the bottom of the stack and place the stack UPSIDE DOWN in the 37°C incubator over night. Kathryn Bizier, Malden High School Question: Why do the plates need to be grown at 37°C? 13. Predict: What do you expect to grow on each of the plates? LB agar LB agar + ampicillin Bacteria + GFP DNA Do you expect to see any difference in growth? Pre-­‐Lab Questions for Day 2 1. What purpose is ampicillin serving in this experiment? 2. If the plasmid DNA hadn’t been added, what do you think bacterial growth would have looked on both plates? Day 2 – Observations 1. What do you see on both of your plates? Draw (or take a picture) of your results below? 2. Now look at both plates using the UV light. What do you see? Kathryn Bizier, Malden High School LB agar Lab agar + ampicillin 3. Fill in the data table below Type of agar plate What kind of growth(s) do you see (phenotype)? What does this tell you about the DNA (genotype)? Analysis Questions 1. What does the plasmid DNA allow the bacteria to do? 2. The GFP gene came from jellyfish that causes jellyfish to glow. What you looked at the cells with UV light, did your bacteria glow? Explain your results. Kathryn Bizier, Malden High School Purification of Recombinant Green Fluorescent Protein Adapted from: web.pdx.edu/~yanm/pGLOlab INTRODUCTION In the previous lab you performed a genetic transformation of E. coli bacterial cells. The results of this procedure were colonies of cells that fluoresced when exposed to ultraviolet light. This is not a normal phenotype (characteristic) for E. coli. After determining that the pGLO plasmid DNA was not responsible for the fluorescence under the UV light, you concluded (hopefully!) that it was not the plasmid DNA that was fluorescing in response to the ultraviolet light within the cells, but a protein that the new DNA produces within the cells. In this lab, one white colony from an LB plate and one green colony from an LB +amp plate will be grown in separate liquid cultures. Since it is hypothesized that the cells contain the Green Fluorescent Protein, and it is this protein we want to produce and purify, we first need to grow up large amounts of the cells so that we can analyze them for their protein contents. Once we have grown a large enough number of cells, we can then perform our protein analysis. Because the GFP synthesized by the E. coli is inside of the cells, before GFP can be isolated you will first need to break open the cells to release it into solution. There are a number of methods for breaking open bacterial cells, and you will use treatment by the enzyme lysozyme followed by freezing on dry ice. Lysozyme is an enzyme that functions to degrade the bacterial cell wall by cleaving polysaccharide (sugar) residues in the cell walls (bacterial cell walls are composed of a network of sugar polymers cross-­‐linked by short chains of amino acids. This structure is known as peptidoglycan). The subsequent freeze-­‐thaw step used aids in the complete disruption of the cell wall and internal membrane. Complete disruption or "lysis" of the cell releases soluble components inside the cell, including GFP. Lysozyme is naturally found in, among other places, human tears, where it acts as a bactericidal agent to help prevent bacterial eye infections. Lysozyme gets its name from its natural ability to "lyse" bacteria. The lysozyme that you will use comes from egg whites. The bacterial lysate generated contains a mixture of GFP and other bacterial proteins. Your goal is to separate and purify GFP from these other contaminating bacterial proteins. Proteins are long chains of amino acids, some of which are very hydrophobic or "water-­‐hating". GFP has many patches of hydrophobic amino acids, which collectively make the entire protein hydrophobic. Moreover, GFP is much more hydrophobic than most of the other bacterial proteins. We can take advantage of the hydrophobic properties of GFP to purify it from the other, less hydrophobic (more hydrophilic) bacterial proteins using a technique known as chromatography. Chromatography is a powerful method for separating proteins and other molecules in complex mixtures. In chromatography, a column is filled with microscopic spherical beads, often called a gel or matrix. A mixture of proteins in a solution passes through the column by moving downward through the gel. Gels are available with a variety of different properties, including charge (positive and negative) for carrying out separations based on the differences in charge of the proteins in a sample (ion-­‐exchange chromatography), pore size, for carrying out separations based on the size of the proteins in a sample (size-­‐exclusion chromatography), and hydrophobicity, for carrying out separations based on differences in the hydrophobicity of proteins in a sample (hydrophobic interaction chromatography, or HIC). You will be using a column filled with beads that are very hydrophobic, and thus will be performing HIC. When the bacterial lysate is applied to the column, the Kathryn Bizier, Malden High School hydrophobic proteins in the sample will stick to the beads while hydrophilic proteins in the mixture will pass through. This interaction is strengthened by applying the sample to the column in a buffer containing a high concentration of salt. When the salt concentration is decreased, the hydrophobic proteins will no longer stick to the gel as strongly and will drip out the bottom of the column in a purified form. EXPERIMENTAL PROCEDURE The day before the lab: 1. Take the plates out of the 37 °C incubator. 2. Fill two 15 mL test tubes (that can be closed) with LB. Label one tube +GFP and the other tube –GFP. 3. Using a new sterile loop for each plate, pick up one colony from each plate, and innoculate the the corresponding tube. 4. The tubes should be placed into the 37 °C incubator over night. 5. Check to make sure that the tubes have a cloudy appearance the next day which indicates that bacterial growth occurred. Lysing of bacteria & protein chromatrography Solutions and Materials Needed Solutions: TE buffer: 10 mM Tris, 1 mM EDTA (pH 8.0) Equilibration buffer TE plus 2.0 M NH4SO4 (pH 8.0) Binding buffer TE plus 4.0 M NH4SO4 (pH 8.0) Wash buffer: TE plus 1.3 M NH4SO4 (pH 8.0) Lysozyme Materials: Microcentrifuge tubes UV Light Transfer Pipettes Micropipettes (p20, p1000) Chromatography Columns Micropipette Tips Microcentrifuge Waste Beaker Dry Ice Test Tubes Test Tube Racks Lab Markers Protocol 1. Using a marker, label one new microtube with your name and lab period. 2. Obtain the two liquid cultures from your teacher and observe them in normal room lighting and then with the UV light. Note any color differences that you observe. Using a pipette, transfer 1.7 mL of the (+) liquid culture into the labeled microtube, then cap it. Set aside the (-­‐) culture for disposal. Question: Did you observations match what you predicted you would see? Explain! Kathryn Bizier, Malden High School 3. Spin the microtube for 5 minutes in the centrifuge at maximum speed. Be sure to balance the tubes in the machine. If you do not know how to balance the tubes, DO NOT operate the centrifuge without asking for help first. Centrifugation is a good way to concentrate a large number of cells. Question: As you spin the cell culture, where would you expect the cells to concentrate, floating at the top of the tube, or bunched together at the bottom of the tube in a pellet? 4. After the bacterial liquid culture has been centrifuged, open the tube and carefully remove the liquid supernatant above the pellet with your pipette. After the supernatant has been discarded, there should be a large bacterial pellet remaining in the tube. 5. Observe the pellet under UV light. Note your observations. Question: What does the pellet look like? What did it do underneath the UV light? Why do you think this happened? 6. Add 250 µl of TE Solution to the tube. Resuspend the bacterial pellet thoroughly by rapidly pipetting up and down with the pipette. Note, do NOT bubble the solution by allowing air into your pipette. Continue until all of the cells are resuspended and there are no clumps of bacteria. 7. Add 10 µl of a solution of lysozyme to the resuspended bacterial pellet. Cap and mix the contents by flicking the tube with your index finger. The lysozyme will start digesting the bacterial cell wall. Observe the tube under the UV light. Question: What do you see under the UV light after you’ve added the lysozyme? Do you see anything interesting or unexpected? 8. After ~10 minutes place the tube in dry ice for ~5 minutes so that the solution completely freezes. BE CAREFUL, do NOT touch the dry ice with your hands! 9. Remove your microtube from the dry ice and thaw it out using hand warmth. Place the tube in the centrifuge and pellet the insoluble bacterial debris by spinning for 10 minutes at maximum speed. 10. Label a new microtube with your group’s initials. 11. While you are waiting for the centrifuge, prepare the chromatography column. Before performing the chromatography, shake the column vigorously to resuspend the beads. Then shake the column down one final time, like a thermometer, to bring the beads to the bottom. Tapping the column on the table-­‐
Kathryn Bizier, Malden High School ` top will also help settle the beads at the bottom. 12. Remove the top cap and snap off the tab at the bottom of the chromatography column. Allow all of the liquid buffer to drain from the column (this will take ~3–5 minutes). Collect the buffer as it drains into a beaker. 13. Prepare the column by adding 2 mL of Equilibration Buffer to the top of the column, 1 mL at a time using your pipette. Drain the buffer from the column until it reaches just above the top of the white column bed. Cap the top and bottom of the column. 14. After the 10 minute centrifugation, immediately remove the microtube from the centrifuge. The bacterial debris should be visible as a pellet at the bottom of the tube. The liquid that is present above the pellet is called the supernatant. Examine the tube with the UV light. Question: What do you see under the UV light? Where does it show the GFP protein is currently located? In other words, is it in the pellet currently, or in the supernatant? How do you know this? 15. Pipette 250 µl of the supernatant into a new microtube. Kathryn Bizier, Malden High School 16. Pipette 250 µl of Binding Buffer to the microtube containing 250 µl of the supernatant. 17. Obtain 3 test tubes and label them 1, 2, and 3. Place the tubes in a rack. Remove the cap from the top and bottom of the column and let it drain completely into a liquid waste container (an extra test tube will work well). When the last of the buffer has reached the surface of the HIC column bed, gently place the column on collection tube 1. Do not force the column tightly into the collection tubes—the column will not drip. 18. Predict what you think will happen for the following steps and write your predictions, along with your actual observations in the data table on the next page. 19. Carefully load 250 µl of the supernatant with Binding Buffer onto the top of the column by resting the pipette tip against the side of the column and letting the supernatant drip down the side of the column wall. Examine the column using the UV light. Note your observations in the data table. Let the entire volume of supernatant flow into tube 1. 20. Transfer the column to collection tube 2. Carefully add 250 µl of Wash Buffer and let the entire volume flow into the column. As you wait, predict the results you might see with this buffer. Examine the column using the UV light and list your results in the data table. 21. Transfer the column to tube 3. Carefully add 750 µl of TE buffer (Elution Buffer) and let the entire volume flow into the column. Again, make a prediction and then examine the column using the UV light. List the results in the data table. Kathryn Bizier, Malden High School 22. Examine all of the collection tubes using the UV lamp and note any differences in color between the tubes. Label the tubes (name and lab section) and give them to your lab teacher to be frozen until the next class so that they can be exmained further. Data Table Analysis Questions Kathryn Bizier, Malden High School 1. What color was the bacterial pellet after centrifuging the intact cells? After centrifuging the lysed cells? What color were the supernatants? What does this tell you? 2. Using the data table above, compare how your predictions matched up with your observations for each buffer. In other words, did your data match your predictions? Why or why not? a. Binding Buffer. b. Wash Buffer. c. Elution Buffer. 3. Based on your results, explain the roles or functions of these buffers. a. Equilibration Buffer -­‐ b. Binding Buffer -­‐ c. Wash Buffer -­‐ d. TE (Elution) Buffer -­‐ 4. Were you successful in isolating and purifying GFP from the cloned bacterial cells? Identify the evidence you have to support your answer. Kathryn Bizier, Malden High School Name: Date: Period: pGLO Plasmid Worksheet 1. How many base pairs long is the pGLO pasmid? About how many base pairs long is the GFP gene? 2. How do we abbreviate the origin of replication within the plasmid? What purpose does this serve? 3. You want to cut this plasmid into 3 pieces. 1 piece needs to contain the GFP gene & promoter, and should be 1,038 base pairs long. One pieces should contain the ampicillin resistance gene & origin of replication, and should be 2,462 base pairs long, and the third piece should be 1,871 base pairs long. Which restriction enzymes should you use? To start you off, here is one of the restriction enzymes that should be used: SanDI. 4. What is the purpose of the arabinose promoter? 5. How do we abbreviate the ampicillin resistance gene, and what purpose does this serve? Below you will find the sequence and cut sites for the GFP gene. Please use that information to answer the questions that follow. Kathryn Bizier, Malden High School Kathryn Bizier, Malden High School 1. What are the first 3 amino acids in the GFP protein chain? 2. What enzyme would you use to cut out the GFP gene at the beginning of the sequence? 3. What enzyme would you use to cut out the GFP gene at the end of the sequence? 4. What enzymes would you not want to use to cut out GFP, and why? 5. What purpose does the arabinose promoter serve? Now go to the following website: https://www.neb.com/products/restriction-­‐endonucleases Scroll down to the list under “Convenience” and open the 276 restriction endonucleases list. You are going to click on a few of the different restriction enzymes that they have listed here to find out a little information about each of them. Just click on the name for each RE and answer the questions that follow. There are some very helpful and informative icons that look like below: Restriction Enzyme: BamH1 1. What is its optimum incubation temperature? 2. Can it be used in overnight incubations? Kathryn Bizier, Malden High School 3. What type of cut does BamH1 make? 4. What temperature should it be stored at? 5. What concentration does BamH1 come in? 6. Can it be inactivated by heat? Restriction Enzyme: EcoR1 1. Please draw is cut sequence, and indicate where cuts occur, below. 2. At what temperature can EcoR1 be inactivated at? 3. What pH should this restriction enzyme be kept at? 4. How quickly can EcoR1 cut up a DNA sample? Now do a little research, and see if you can find an enzyme that functions at a temperature other then body temperature. Write the enzyme below, and give some additional information about that particular enzyme. Kathryn Bizier, Malden High School