Lab 8: Bacterial Transformation with pglo for Protein Production

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1 OBJECTIVES: Lab 8: Bacterial Transformation with pglo for Protein Production Describe the principles of chromatography. Explain the procedure for the production of engineered proteins. Isolate the Green Fluorescent Protein (GFP) from transformed E. coli. Review the process of transcription and translation. Know the terms used in this lab including chromatography, pellet, supernatant Answer the questions posed in this lab. PRELAB: Review Transcription and Translation if needed Read this lab and write out your protocol in your lab notebook. Answer the following questions: 1. What types of molecules do you expect to be released when your bacterial cells are lysed? Provide at least three good examples! 2. In your own words, define column chromatography. What will this technique do for us in this experiment? 3. What does the Binding Buffer do in our experiment? 4. What is the purpose of the TE (Elution buffer) in our experiment? INTRODUCTION: In last week s lab we successfully transformed bacteria with a jellyfish gene. Recall that we transformed them by introducing a plasmid carrying several specific genes, including the gene for Green Fluorescent Protein (GFP). The bacteria then transcribe and translate these genes to generate the protein encoded by the plasmid DNA. In industry, such transformations are done routinely to generate engineered proteins. For instance, many individuals with diabetes no longer produce the hormone, insulin (a protein). These individuals inject recombinant insulin made in bacteria to manage their diabetes. Whether you re producing a green fluorescent protein for fun, or making insulin to save lives, the basic procedures are the same. You must first grow up large volumes of bacterial cells producing your protein of interest. These bacterial cells can then be lysed (broken open), allowing the protein of interest to be purified from all the other bacterial proteins present in the cells. This will be the goal for this portion of lab. Purifying your target protein is no simple task. To do this, we ll use a technique called chromatography. Chromatography is a powerful technique for separating proteins in a complex mixture; it allows us to separate our protein of interest, GFP, from the complex mixture of molecules found inside the bacterial cells. The technique requires that you have some knowledge of a molecule s chemical properties. In our case we re fortunate in that GFP is

2 extremely hydrophobic compared to most other bacterial proteins. Thus we can use the interaction of GFP with a chromatography matrix to select it out of the mixture of bacterial molecules. To do this we ll run our protein mixture across a bed of microscopic beads in a cylinder or column. These beads form a matrix through which proteins must pass before being collected. This matrix has an affinity for GFP, but not most of the other bacterial proteins. Thus GFP will stick to the column while other molecules flow through. More precisely, in a high-salt environment, the three dimensional structure of GFP changes slightly, exposing hydrophobic regions of the protein. Thus in the presence of salt GFP will stick to the beads. When the high salt environment is replaced by a low salt solution, GFP reverts to its original conformation and drips out the bottom of the column. As before, because this protein glows, we ll be able to follow the success of our technique at each step. DETAILED PROTOCOL- PART I: 1. Examine your transformation plates with the UV light. Identify several green colonies that are not touching other colonies on the LB/amp/ara plate. Mark their locations using a sharpie. (Label the outside of the plate!) 2. Obtain a culture tube containing liquid LB with ampicillin and arabinose. Label your tube. Use a sterile loop to gently scrape off a single green colony. Immerse this loop in the tube and twirl gently to disperse the colony. 3. Cap the tube and place it in a shaking incubator (if available) at 32 C for hours. If tubes are not in a shaking incubator, lay them horizontally and shake periodically when possible to maximize oxygen levels in the media.

3 OVERVIEW OF METHODS:

4 DETAILED PROTOCOL- PART II: A. Concentration and Lysis of the Bacteria 1. Label one microtube. Remove your liquid culture from the shaking incubator and observe with the UV light. Using a sterile pipette, transfer 2 ml of the liquid culture into the microtube. Spin the microtube for 5 minutes at maximum speed in the microcentrifuge. 2. When the spin is complete, note that you have a solid pellet at the bottom of the tube (the bacterial cells) and a clear supernatant, or fluid, above it. Pour out the supernatant and examine the pellet under UV light. 3. Using a rinsed pipette, add 250 ul of TE solution to the tube. Resuspend the pellet thoroughly by rapidly pipetting up and down several times. 4. Using a rinsed pipette, add 1 drop of lysozyme to the resuspended bacterial pellet to begin the enzymatic digestion of the bacterial cell wall. Mix the contents gently by flicking the tube. Observe under UV light. 5. Place the microtube in the freezer for 10+ minutes. Freezing will cause the bacteria to rupture completely. Complete lysis releases all the soluble contents of the cell, including the GFP protein. B. Protein Purification and Column Preparation 1. Remove the microtube from the freezer and thaw using hand warmth. Place the tube in the centrifuge and pellet the insoluble bacterial debris by spinning for 10 minutes at maximum speed. 2. While your tube is spinning, prepare the chromatography column. Remove the cap and snap off the bottom of the column. Allow all of the liquid buffer to drain from the column (3-5 minutes). 3. Prepare the column by adding 2 ml of Equilibration Buffer to the top of the column. When adding anything to your column, be sure to pipette slowly, allowing the solution to drain down the side of the tube, minimizing the disturbance to the top of the column. 4. After the 10 minute spin, remove your tube from the centrifuge and examine it with the UV light. Using a new pipette, transfer 250 ul of the supernatant to a new labeled microtube. Add 250 ul of Binding buffer to this new tube.

5 C. Protein Chromatography 1. Label 3 collection tubes 1-3 and place them in the foam rack. Place your equilibrated column in tube 1. When the last of the buffer has reached the surface of the HIC matrix, proceed to the next step. 2. Using a new pipette, carefully and gently load 500 ul of the + supernatant onto the top of the column. After it stops dripping, transfer the column to collection tube 2. Examine both the column and collection tube #1 under the UV light. 3. Using the rinsed pipette, add 250 ul of wash buffer to the column. Allow this volume to flow through the column. After the tube stops dripping, transfer it to tube 3, examining both tube #2 and the column under the UV light. 4. Using the rinsed pipette, add 750 ul of TE Buffer to the column and allow it to drip through. When it is complete, examine the column, and all three collection tubes under the UV light. (Be sure to record these observations in your lab notebook!) 5. Follow your instructor s directions to label and store your eluted protein for our next lab day. DATA AND OBSERVATIONS: Record your predictions, observations, and data in an organized table in your lab notebook! NEXT STEPS: In our next lab exercise, we ll use a special gel to examine a sample of your crude protein (from colonies on your original plates) and your purified protein product (produced today). You ll combine the results of your transformation, today s purification, and our protein electrophoresis for your formal report!