All of the lab investigations in this unit are based on nothing less than authentic Nobel Prize–winning science. Kary Mullis received the Nobel Prize for his discovery of the Polymerase Chain Reaction or PCR. Werner Arbor, Daniel Nathans and Hamilton Smith received the Nobel Prize for their work with restriction enzymes. Stanley Cohen, Paul Berg and Herb Boyer received the highly esteemed prize for making the first recombinant DNA molecule. Actually, the recombinant DNA molecule that students create during this lab series extends beyond the scope of these honored Nobel Laureates original work as it incorporates a gene from a eukaryotic rather than prokaryotic organism into a plasmid. It would be impossible to explain the effect these advancements and breakthroughs have had on the development of biotechnology, medicine, forensic science, and research in general however perhaps by experiencing the same triumphs as these giants the significance will become apparent.
In this laboratory investigation, purify a mutant fluorescent protein from other proteins present in the overnight cell culture from previous lab, Creating a Cell Culture of Ecoli based on our knowledge of how groups of molecules differ from one another and how these differences can be used to effect separation.
The purpose of this lab is to allow the transformed bacteria time to express the mutant protein in an overnight culture and then lyse (broken open) the cells in order to release the newly synthesized protein from the cell so that it can be purified.
I begin this lab with a Guided Simulation activity accompanied by a PowerPoint Presentation in which students must devise and test a method or model for purifying (separating) a protein of interest from other gene products in a simulated cell/protein sample. Using a collection of common craft materials, that possess a range of properties from the ability to float in water to being magnetic, students try to devise a separation technique that will isolate one on the materials.
We then continue by setting the context of the work we will be doing and providing background information as illustrated on SLIDES 12 thru 18 of the AMGEN Recombinant DNA Lab Series PowerPoint Presentation.
At the end of this segment of our prelab discussion I check for understanding using a Pre Lab Quiz as well as visit the community lab area in our laboratory classroom in order to walk students through staging their workspaces or "bench" as shown in this photo. As we move about the community lab area and either stage or review staging that has already been completed, I explain the significance of each piece of equipment and how it will be used in our work. Students record notes from our discussion in their laboratory notebook as oftentimes the information provided appears on our Post Lab Quiz and may be covered in our lab Conclusion Questions.
2mL LB/amp/ara culture of E.coli (See Creating a Cell Culture of E.coli Lab)
Lysozyme (10 mg/mL)
Binding buffer, 4M (NH4)2SO4
Column equilibration buffer, 2M (NH4)2SO4
Column wash buffer, 1.3M (NH4)2SO4
Elution buffer, 10 mM TE
10% Bleach or other disinfectant
TE buffer (same as the elution buffer)
EQUIPMENT AND SUPPLIES
P-200 pipette and tips
P-1000 pipette and tips
Microfuge tube rack
1.5 mL microfuge tubes
6mL waste collection tube
Cell-contaminated waste bag
For the Student:
Part I - Preparation of Overnight Culture
1. Obtain 1 mL LB/amp/ara culture from the instructor.
2. Place this tube into the centrifuge. Important: You or the instructor will need to make certain the tubes have been placed in the rotor in a balanced configuration before the centrifuge is turned on. Centrifuge the microfuge tubes for 5 minutes.
3. After the rotor has stopped, carefully remove your tube to avoid disturbing the cell pellet.
4. Determine the location of the mFP. Is it in the bacterial cell pellet, or in the supernatant (the liquid above the cell pellet)?
5. Once you’ve determined the location of the mFP, carefully decant (pour off) the supernatant into the beaker containing disinfectant. Do this without disturbing the cell pellet.
6. Obtain a second 1 mL aliquot of the overnight culture and repeat steps 3–6.
7. Pick up a tube of “Elution buffer” and “Lysozyme” from your teacher.
8. Invert the microfuge tube containing the cell pellet and, using a small piece of paper towel, try to wick away as much of the liquid as you can from your microfuge tube without 3touching the cell pellet. Discard the used towel in the “cell-contaminated waste” bag.
9. Using the P-1000 pipette (set at “0-2-5”) and a clean tip, transfer 250μL of elution buffer to the cell pellet. Close the cap tightly.
10. Resuspend the cells by dragging the tightly capped microfuge tube briskly across the surface of the microfuge tube rack. You may need to do this several times to resuspend the cells. Examine the tube carefully to make certain there are no visible clumps of cells.
11. Using the P-200 pipette (set at “0-4-0”), transfer 40μL of lysozyme to the resuspended cells. Lysozyme is an enzyme that digests the bacterial cell wall. This enzymatic digestion of the cell wall greatly weakens the cell, and the cells will begin to lyse (break open). Finger vortex or drag the microfuge tube across the surface of the tube rack to mix thoroughly. If time permits, incubate samples at room temperature for 60 or more minutes before freezing.
12. Make certain that you have labeled this tube with your group number and class period. Give the tube to your teacher. These cells will be placed into the freezer following the incubation.
13. Cells can remain frozen, at –20°C, until the next lab session.
Part II - Purification of the mFP
1. Centrifuge the cell lysate for five minutes to pellet the cell debris. You or your teacher will need to check the rotor to be certain it is balanced before closing the lid and spinning. Balancing these tubes before centrifugation is very important.
2. After centrifugation, pick up your microfuge tube. Examine the microfuge tube. Where is the mFP: supernatant or cell pellet?
3. Without disturbing the cell debris pellet, carefully remove 200μL of supernatant using the P-200 pipette (set at “2-0-0”) and a clean tip. Do this without transferring any cell debris. If you dislodge the debris pellet, you will have to centrifuge the tube again. Dispense the 200μL of clean, debris-free supernatant into a 1.5 mL microfuge tube labeled “super”(one of your group members should have labeled this tube).
4. Replace the pipette tip on the P-200 and add 200μL of binding buffer to the supernatant you dispensed in the tube labeled “super.” Mix the binding buffer with the supernatant by gently pumping the solutions in and out using this pipette.
5. Using the p-1000 pipette (set at “0-4-0”) and a clean tip, add 400μL of this solution, mFP supernatant/binding buffer, to the prepared column using the same pipette you used to mix the solutions. Do this without disturbing the surface of the resin bed by dispensing the solution down the side of the column.
6. Without allowing the column to run dry, open the stopcock and allow the solution in the column to drain into the waste collection tube. Leave about 1or 2 mm of buffer above the resin bed.
The overnight cell culture of Ecoli expressing the rfp gene will be a purified sample of red fluorescent protein at the end of our investigation. This is great time to introduce the idea of activated or "folded" proteins in connection with the fluorescence that is observed in the sample when observing under UV light. I use toys call tangles (small and large) to introduce the idea of protein only becoming active once the have folded into their tertiary structure!
To end this lab investigation students individually answer the Lab 7 Conclusion Questions in preparation for a whole group discussion on the topics presented in this experiment.