Bacterial Transformation and Protein Purification

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1 Bacterial Transformation and Protein Purification Group 4 Natalie Beale Gregory A. Pate Justin Rousseau Dohee Won

2 Introduction The purpose of this experiment is to perform a genetic transformation and a protein purification. The process of genetic transformation is performed by the introduction, uptake and expression of foreign DNA in a new cell. The successful transformation of a cell is usually identified by the expression of a new trait. In this experiment the new trait that is expressed is the pglo plasmid that codes for Green Fluorescent Protein (GFP) and beta-lactamase which will provide a transformed E. coli cell with ampicillin antibiotic resistance. The transformed cells will then be selectable by antibiotic resistance and GFP which will cause them to glow green under ultraviolet light. Expression of GFP can be activated by growing the transformed cells in a nutrient media containing the simple sugar arabinose. Genetic transformation is used as an inexpensive and simple method for producing large amounts of a desired protein. To purify the GFP we will perform two separations by centrifugation, a lysozyme digestion of the bacterial cell wall to release GFP, and protein chromatography. The beads in the protein chromatography column have an affinity for GFP through hydrophobic interactions. By manipulating the salt concentration of the elution buffer we will purify GFP.

3 Part I: Bacterial Transformation 1. Prepare nutrient agar A. Add 500 ml of distilled water to 1000 ml Erlenmeyer flask B. Add the entire contents of the Luriana Bertani (LB) agar packet to water C. Place Erlenmeyer flasks in autoclave and autoclave for 15 minutes at 121.5ºC D. Place Erlenmeyer Flasks in 50ºC water bath until cool (Can touch with hand for 10 seconds). E. While waiting, mark 16 plates LB, 16 plates LB/amp and 8 plates LB/amp/ara 2. Prepare arabinose and ampicillin A. Prepare Arabinose 1. Add 3ml of transformation solution to vial of dry arabinose 2. Mix with vortexer 3. Allow 10 minutes for arabinose to dissolve completely B. Prepare ampicillin 1. Add 3ml of transformation solution to vial of dry ampicillin 3. Pour Agar Plates A. Pour 16 LB plates 1. Stack LB plates 6 high and have one person tilt lids upward, while other person quickly pours agar into plates. If bubbles appear, can quickly run plate over flame of Bunsen burner. 2. Add contents of one Erlenmeyer flask to the other B. Pour 16 LB/amp plates 1. Add rehydrated ampicillin to remaining LB agar and swirl to mix 2. Pour plates in similar fashion as LB plates C. Pour 8 LB/amp/ara plates 1. Add rehydrated arabinose to remaining agar and swirl to mix 2. Pour plates in similar fashion as LB plates 4. Rehydrate bacteria A.Using a sterile pipette, rehydrate the lyophilized E.coli HB101 by adding 250 μl of transformation solution directly to the vial B. Recap the vial and allow the cell suspension to stand at room temperature for 5 minutes C. Then shake to mix, before streaking LB starter plates D. Store in refrigerator until use. 5. Streak starter plates to produce single colonies on agar plates A. Insert a sterile inoculation loop into the rehydrated E. coli bacterial culture. Insert the loop straight into the vial and do not tilt the vial. Remove the loop and streak the plates. Streaking takes place sequentially in four quadrants. The first streak is to just spread the cells out a little. Go back and forth with the loop about a dozen times in each of the small areas. In subsequent quadrants the cells become more and more dilute, increasing the likelihood of producing single colonies.

4 B. For subsequent streaks, the goal is to use as much of the surface area of the plate as possible. Rotate the plate approximately 45 degrees (so that the streaking motion is comfortable for your hand) and start the second streak. Do not dip into the rehydrated plasmid a second time. C. Rotate the plate again the repeat streaking. D. Rotate the plate for the final time and make the final streak. Repeat steps A-D with the remaining LB plates for 3 workstations. Use the same inoculation loop for all plates. When you are finished with each plate, cover it immediately to avoid contamination. E. Place the plates upside down inside the incubator overnight at 37 C. 6. Prepare pglo plasmid A. Using the micropipetter add 250 μl of transformation solution into the vial of lyophilized pglo plasmid DNA. B. Store in refrigerator until use 7. Aliquot solutions A. Aliquot 1 ml of transformation (CaCl 2 ) solution and and 1 ml of LB broth into separate color-coded tubes B. Refrigerate until use if aliquotted one day prior to lab

5 Transformation Procedures 1. Label one, closed micro test tube +pglo and another pglo. Place them in foam tube rack 2. Open tubes and using the micropipetter, transfer 250 μl of transformation solution to each microtube 3. Place the tubes on ice 4. Using a sterile loop, pick up a single colony of E. coli from your starter plate. Place the loop into transformation solution of micro test tube +pglo and mix. (Ensure that there are no floating chunks). Repeat this step with a new sterile loop and mix in micro test tube pglo. 5. Examine the pglo plasmid DNA under UV lamp. Note observations. Immerse sterile loop into plasmid DNA stock tube and withdraw a loopful. Mix loopful into micro test tube with +pglo. DO NOT MIX IN MICRO TEST TUBE pglo 6. Incubate the tubes on ice for 10 minutes. Tubes must be pushed all the way down in the rack so the bottom of the tubes stick out and make contact with the ice. 7. While tubes are on ice, label the 4 agar plates as follows: LB/amp (+pglo), LB/amp/ara (+pglo), LB (-pglp) and LB/amp (-pglp)

6 8. Heat shock the tubes. Place foam rack in 42 C water bath for 50 seconds. After this, place on ice for 2 minutes. 9. Remove rack from ice and place on counter. Transfer 250 μl of LB broth to each tube and incubate at room temperature for 10 minutes. 10. Mix tube contents by tapping. Then, using a new sterile pipette for each tube, extract 100 µl the transformation and control suspension onto the appropriate plates. 11. Spread the suspensions evenly around the surface of the agar by quickly skating the flat surface of a new sterile loop back and forth across the plate surface. 12. Place them upside down in the 37 C incubator.

7 Part II: Protein Purification Protein Purification Procedures 1. Make LB/amp/ara culture tubes A. Rehydrate arabinose and ampicillin with TE solution B. Add 0.5 ml arabinose and 0.5 ml ampicillin to 55 ml of pre-made LB broth C. Aliquot 2 ml of LB/amp/ara broth to 16 culture tubes 2. Create LB/amp/ara culture tubes A. Using a sterile loop, remove a single colony from the LB/amp/ara plate and add it to one culture tube, label tube + B. Using a sterile loop, remove a single colony from LB/amp plate and add it to the other culture tube and label the tube - C. Place culture tubes in incubator at 32 C for 2 days 3. Label one microtube +. Observe your liquid culture with the UV light. Note any color differences between the two cultures. Transfer 2ml of + culture into + microtube, and 2ml of - culture into - microtube. 4. Centrifuge microtubes at maximum speed for 5 minutes 5. Pour out supernatant and view with UV light. 6. Add 250 μl of TE solution to the tube and resuspend pellet by rapidly pipetting up and down several times

8 7. Add 1 drop of lysozyme to resuspended bacterial pellet. Mix by gently flicking the tube. Incubate the tube at 37 C for 5 minutes. 8. Oberve with UV light 9. Place the tubes in Nitrogen bottle for 5 minutes. 10. Remove tubes from Nitrogen bottle and warm with hands until thawed. Centrifuge at maximum speed for 10 minutes 11. While tubes are spinning, prepare chromatography column. Remove cap and snap off the bottom from the prefilled HIC column. Allow all of the liquid buffer to drain from the columns (~3-5 minutes). 12. Prepare the column by adding 2 ml of Equilibration buffer to the top of the column and drain the buffer to the 1 ml of mark on the column. 13. After centrifugation, examine tubes with UV light. Transfer 250 μl of + supernatant into a new tube labeled +, and - tube, too. 14. Transfer 250 μl of binding buffer to the + supernatant, and - supernatant. 15. Sit for 5 minutes. 16. Label collection tubes 1-3 and place in foam racks. Remove caps from top and bottom of column and place in collection tube 1. When the last of the buffer has reached the surface of the HIC matrix, move to next step

9 17. Carefully load all liquid of + supernatant onto the top of the column. Hold the micropipette tip to side of column and allow to gently drip down side of column. After dripping has stopped, transfer column to tube Add 250 μl of wash buffer to column. Examine collection tube with a UV light. After dripping stops, transfer column to tube Add 750 μl of TE buffer and allow the volume to flow through column. Examine with a UV light. 20. Examine all 3 collection tubes with a UV light.

10 Review Questions 1. How does the pglo plasmid appear under UV light? 2. On which of the plates would you expect to find bacteria most like the original untransformed E. coli colonies you initially observed? 3. How did the two culture tubes appear with the blacklight? 4. What was the effect of the buffers? Equilibration Binding Wash Elution 5. Note your observations of the collection tubes.

11 Analysis of the Results 1. Which of the traits that you originally observed for E. coli did not seem to become altered? 2. Of the E. coli traits you originally noted, which ones now seem to be significantly different after performing the transformation procedure? 3. If the genetically transformed cells have acquired the ability to live in the presence of the antibiotic ampicillin, what can be inferred about the other genes on the plasmid that were involved in your transformation procedure?

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