Transforming Escherichia coli with a Recombinant Plasmid
Lesson 4 of 9
Objective: Students will be able to use a technique called transformation to get their engineered recombinant plasmids into bacterial cells which will express novel genes inherited during this process.
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, students use the process of bacterial transformation which entails the taking up of foreign pieces of DNA, such as a plasmid, into a bacterial cell to confirm the inclusion of the rfp gene in the pARA-R plasmid that they genetically engineered in the previous lab, Producing a Recombinant Plasmid, pARA-R.
The purpose of this lab is to get the recombinant plasmids engineered by students into bacterial cells (Ecoli) so that they can express the newly incorporated rfp gene and make the mutant fluorescent protein.
I begin this lab by setting the context of the work we will be doing and providing background information as illustrated on SLIDES 8 thru 11 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 will be covered in our lab Conclusion Questions.
LIG tube (recombinant plasmids from Producing a Recombinant Plasmid pARA-R Lab)
100 uL of competent cells (LMG) or Ecoli Starter Plates
350 uL of LB broth (sterile)
Crushed ice (in a styrofoam cup)
Agar plates, sterile
1 LB, 1 LB/amp, 1 LB/amp/ara
EQUIPMENT & SUPPLIES
P-20 micropipette and tips
P-200 micropipette and tips
42C water bath
1 pack cell spreaders (shared)
Plastic microfuge tube rack
1.5 mL microfuge tubes
For the Student:
1. Pick up two clean microfuge tubes. Label one “P+” and the other “P-.”
2. Pick up a Styrofoam cup with crushed ice and place one tube containing 100 μL of competent cells into the ice. It’s important that the cells remain at 0°C. Also, place your P+ and P- tubes into the ice.
3. Pick up your ligated plasmids from the microfuge tube rack labeled “LIG tubes.” Your “LIG” tube should be labeled with your group number and class period.
4. Set the P-200 pipettor to 50 μL (set to “0-5-0”) and place a clean tip onto its barrel. Very carefully resuspend the cells by gently pumping the cells in and out two times. Hold the tube by the upper rim to avoid warming the cells with your fingers.
5. Aliquot 50 μL of the resuspended cells into the prechilled P+ and P- microfuge tubes. Immediately return the aliquoted cells to the wet ice. Hold the tubes by the upper rim to avoid warming the cells with your fingers.
6. Using the P-20 pipette, add 10 μL of your ligated plasmid to the tube labeled “P+.” Gently mix the plasmid with the cell suspension by pumping the cell suspension two times. Immediately return the P+ tube into the ice. Do not add plasmid to the P– tube. The cells in this tube will serve as the “plasmid control.”
7. Keep the cells in ice for 15 minutes.
8. While the cells are incubating in ice, obtain the following: One each of these agar plates: LB, LB/amp (LB + ampicillin) and LB/amp/ara (LB + amp + arabinose)
9. Label the bottoms (plate containing the agar) of all three plates with your group number and class period. Write small and on the edge of the plate. Then divide the LB and amp plates down the middle using two lines. Label one half of each plate “P+” and the other half with a “P-.” See the diagram below. Do not divide the ara plate.
10. Following the 15-minute incubation in ice, carry the ice cup containing the cells to the 42°C water bath. Take the tubes from the ice and hold them in the water bath for 45 seconds. After the 45-second heat shock, place them back into the wet ice immediately for at least one minute.
11. After one minute, use the P-200 pipette to add 150 μL (set to “1-5-0”) of LB broth to the P- tube. Cap the tube and gently flick the lower portion of the tube two or three times to mix.
12. Use a new tip and transfer 150 μL of LB broth to the P+ tube. Close the cap and gently flick the tube to mix.
13. Obtain one package of sterile cell spreaders from your teacher. You are now ready to spread your bacterial cells onto the sterile agar plates.
14. Using the P-200 pipette (set to “0-5-0”), gently pump the pipette two or three times to resuspend the cells then aspirate 50 μL of cells from the P- tube. Open the lid from the LB plate like a “clamshell.” Dispense these cells on the half of the plate marked “P-.” Close the lid.
15. Resuspend the cells by gently pumping the pipette then aspirate a second 50 μL aliquot for the LB/amp plate. Remember, you want to deposit the P– cells on the half of the plate you labeled “P-.” Cover the plate.
16. Open the package of sterile cell spreaders at the end closest to the spreader handles. You will share this package with another group. Remove only one spreader, keeping the others sterile. Hold the spreader by the handle and do not allow the bent end to touch any surface, as this will contaminate the spreader. Close the package to avoid contaminating any of the other spreaders.
17. Open the lid to the LB plate, like a clamshell, and gently using a light, gliding motion spread the cells across the surface of the agar, keeping the cells on the P– side of the plate. Try to spread them evenly and along the sides of the plate as well. Carefully spread the P- cells on the LB/amp plate using the same spreader and technique. Place the used spreader into the biohazard bag.
18. Repeat steps 14 thru 17 to inoculate the LB and LB/amp plates with the P+ culture. Be certain to use the “+” pipette and a new spreader to avoid contamination.
19. Now you’re ready to inoculate the LB/amp/ara plate! Using the P-200 pipette (set to “1-0-0”), transfer 100 μL of the P+ culture onto the surface of the LB/amp/ara plate. Deposit the 100μL of cells on several areas across the agar surface rather than a single spot. Lift the lid, clamshell style, and spread the cells evenly over the surface of the plate. Gently rotate the plate beneath the P+ spreader so that the cells can be spread over the entire surface of this plate. Try to get the cells spread along the wall of the plate as well. Cover the plate when finished.
20. Allow all three plates to sit right-side up for five minutes.
21. Using colored tape, tape all three plates together and place them in the incubator, gel-side up. Be certain that you have clearly labeled your plates with your group number and class period. You can mark the tape to help you find them for the next lab.
22. Discard cell-contaminated waste: spreaders, cell tubes, pipette tips, by placing them into the cell-contaminated waste bag provided by your teacher.
The following photo illustrates the expected result for this laboratory investigation. Please make note of the manner in which students labeled the plates and the information that they provided. This is a MORE than typical result however I believe that it still holds value as both a training tool before the lab and perhaps as a control in which to compare the results received by instructors and their students at the conclusion of the lab.
To conclude this lab students individually complete the Lab 5 conclusion questions and prepare to share their insights during a whole group discussion. Since this lab includes the process of transformation which is a core practice in confirming the creation of genetically engineering plasmids, students complete an exit ticket in the form of a mock lab memo in which they have to explain to their pretend "employees" how to complete a bacterial transformation assay! They must also explain to their staff how to interpret the Lab 5 Results received and provide evidence for their interpretation!