7.3: Transforming Bacteria with Plasmids

In this laboratory you will transform E. coli bacteria with plasmids. Using E. coli bacteria that have been pretreated with calcium chloride, you will divide the bacteria into two groups: a control group to which no plasmid is added, and a treatment group to which you add the plasmids. After heat-shocking both groups of cells, you will grow them under several different conditions: 

• The control group is grown on nutrient agar (a medium that supports bacterial growth). 

• The control group is grown in the presence of nutrient agar and the antibiotic ampicillin. 

• The treatment group is grown in the presence of nutrient agar. 

• The treatment group is grown in the presence of nutrient agar and the antibiotic ampicillin. 

• The treatment group is grown in the presence of nutrient agar, ampicillin, and an inducer (such as IPTG). 

By examining the growth of bacteria under these conditions, you can verify that your procedure worked, and you can identify the bacteria transformed with
the plasmid that you added to the bacteria. How will you know if you are successful? In the examples we have recommended, the recombinant bacteria will have a new and highly visible trait: It will now produce colored protein, which makes the cells themselves colored! 


The relevant components of this plasmid are the gene for the colored protein, the inducible promoter, and the ampicillin resistance gene (ampR). The ampR gene confers resistance to the antibiotic ampicillin. (Biotechnologists call these genes selectable markers because only bacteria having the gene will survive in the presence of an antibiotic.)  If the inducer is present in the bacteria, the promoter will be “turned on” so RNA polymerase can transcribe the gene of interest. This will allow protein to be produced.  

Prelab Questions

Discuss the following questions with your group and be prepared to share your answers with the class. 

• Ampicillin is an antibiotic that kills bacterial cells by disrupting the formation of cell walls. However, the  plasmid has the ampicillin resistance gene, which produces a protein that breaks down ampicillin. What is the purpose of growing bacteria that have been transformed in the presence of ampicillin? 
• What will happen when bacterial cells that contain the plasmid are not given the chemical inducer? 
• In the lab, you will add samples of the control group (no plasmid) P– and the treatment group (with plasmid) P+ to plates that contain various combinations of nutrient agar (NA), ampicillin (AMP), and the chemical inducer (IND).

What are your predictions for the growth you would expect for each combination? Fill in Table 13.1 by indicating whether you predict growth would occur or not. 

• Read through the Procedures below and outline the steps, using words and a flowchart in your lab notebook.

Table 13.1 Predictions

Transformation of E. coli
Medium	No plasmid control	Treatment with plasmid
Nutrient Agar
Nutrient Agar + Ampicillin
Nutrient Agar + Ampicillin + Inducer

Materials

Reagents 

• A rack with the following: 
• Microfuge tube of plasmid 
• Microfuge tube of Nutrient Broth (NB) 
• Microfuge tube of 100 μL of chilled competent E. coli cells (CC) 
  • (CC tube must be kept on ice at all times)
• 3 Petri plates with agar: 
  • 1 of NA
  • 1 of NA/amp 
  • 1 of NA/amp/ind 

Equipment and Supplies 

• Styrofoam cup of crushed ice (fill cup first with ice before taking CC tube.)
• 2 1.5-mL microfuge tubes 
• Permanent marker
• Disposable gloves 
• P-20 micropipette 
• P-200 micropipette 
• Tip box of disposable pipette tips 
• Pack of cell spreaders (will be shared among groups) 
• 42°C water bath with floating microfuge tube rack (will be shared among all groups) 
• Timer or clock (will be shared among all groups) 
• Tape (will be shared among all groups) 
• 37°C incubator (will be shared among all groups) 
• Biohazard bag for materials that come into contact with E. coli cells (will be shared among groups) 
• Waste container (will be shared among groups)

Safety

• All appropriate safety precautions and attire required for a science laboratory should be used. Please refer to your instructions. 
• Use caution when handling E. coli bacteria and use aseptic technique. Aseptic technique is a set of procedures that ensure protection of the lab worker and protection of a bacterial sample, which is necessary for the experiment to be successful. Specifically: 
• Do not touch anything that has been or will be in contact with E. coli bacteria. Students handling equipment that comes into contact with bacteria should wear gloves. 
• Try to avoid spills or contamination of surfaces with anything that has been in contact with E. coli bacteria. Immediately inform your teacher if a spill or contamination occurs. 
• When you have finished using microfuge tubes, pipette tips, and cell spreaders, place them immediately into the biohazard bag or waste container, as directed by your teacher. 
• When directed to do so, place your Petri plates back into the original sleeves and in the biohazard bag. 
• Wash your hands well with soap after completing the lab. 

Procedure

1. Check your rack to make sure that you have the reagents listed. 
2. Obtain a CC tube from the ice-filled container, placing it in a Styrofoam cup of ice. The competent cells in this lab must be kept cold—be sure to pick up microfuge tubes by the upper rim to avoid warming the cells. 
3. Label two clean microfuge tubes “P–” and “P+.” 

   

   Figure 13.3. Labeled microfuge tubes

4. Place the P– and P+ tubes in the Styrofoam cup of ice with the CC tube. Bacterial transformation requires sterile techniques. It is essential that these directions be followed precisely. 
5. Using the larger P-200 micropipette, add the competent cells from the CC tube to the P– and P+ tubes: 
   1. Set the P-200 micropipette to 50 μL.
   2. Very carefully, re-suspend the bacterial cells in the CC tube by gently pumping the pipette two times in the solution.
   3. Add 50 μL of CC to each of the empty chilled tubes (P– and P+), holding each tube at its rim to keep it cold, and return each tube quickly to the ice. To avoid contamination, be sure to use a new micropipette tip for each addition. 
6. Using the P-20 pipette, add plasmid to the tube labeled “P+”: 
   1. Set the P-20 micropipette to 10.0 μL. 
   2. Hold the chilled P+ tube by the upper rim and add 10.0 μL of LIG. Mix the solutions by pumping the pipette two times in the liquids, and return the P+ tube to the ice. 
7. Keep the P– and P+ tubes on ice for 15 minutes.  
8. While the cells are on ice, prepare your three agar Petri plates—one plate each of NA, NA/amp, and NA/amp/ind: 
   1. Label the bottom of each plate (the part that contains the agar) with your group number and class period. Write small and on the edge of the plate. 
   2. With the plates closed, draw a line on the NA plate and the NA/amp plate that divides each plate in the middle. Label half of each plate “P–” and the other half “P+.” Label the NA/amp/ind plate “P+.” The plates will be arranged as follows: 

      

      Figure 13.4. Labeled plates

9. Following the 15-minute incubation on ice, carry the P– and P+ tubes (in the cup of ice) to the 42°C water bath. Place the two tubes in the floating microfuge tube rack in the water bath for EXACTLY 45 SECONDS. 
10. After the 45-second heat shock, immediately place the tubes back on ice and leave them there for at least a minute. 
11. Using the large P-200 micropipette, add NB to the P– and P+ tubes: 

    

    Figure 13.5 How to distribute the no plasmid control treatment onto media

    1. Set the P-200 micropipette to 150 μL. 
    2. Add 150 μL of NB to the P– tube. Cap the tube and gently flick two or three times to mix.
    3. Add 150 μL of NB to the P+ tube. Cap the tube and gently flick it two or three times to mix.  
12.  If time permits, allow the cells in the P– and P+ tubes to incubate at room temperature for 15 minutes. 
13. Add cells from the P– tube onto your NA and NA/amp plates: 
    1. Set the P-200 micropipette to 50 μL. 
    2. Gently pump the pipette two or three times in the P– tube to suspend the cells, and load 50 uL of the P- cells.
    3. Open the lid of the NA plate, like a “clamshell,” and add 50 μL of cells from the P– tube to the section marked “P–.” Close the lid. 
    4. Again, gently pump the pipette two or three times in the P– tube to suspend the cells, and load 50 uL of the P– cells. 
    5. Open the lid of the NA/amp plate, like a clamshell, and add 50 μL of cells from the P– tube to the section marked “P–.” Close the lid. 
14. Spread the cells from the P– tube on your NA and NA/amp plates:
    1. Open the package of sterile cell spreaders at the end closest to the spreader handles. Remove only one spreader, and close the package to keep the others sterile. 
    2. Open the lid to the NA plate, like a clamshell, and spread the cells evenly across the entire P– side of the plate by gently moving the spreader across the agar surface. (Keep the cells on the P– side of the plate.) Close the lid. 
    3. Carefully spread the P– cells on the NA/amp plate, using the same spreader and technique. Hold the spreader by the handle and do not allow the bent end to touch any surface, as this will contaminate the spreader. Place the used spreader in the biohazard bag. 
15. Add cells from the P+ tube to your NA, NA/amp, and NA/amp/ind plates: 

    

    Figure 13.6. How to distribute the plasmid treatment onto media

    1. Make sure that the P-200 micropipette is set to 50 μL. 
    2. Gently pump the pipette two or three times in the P+ tube to suspend the cells, and load 50 uL of the P+ cells. 
    3. Open the lid of the NA plate, like a clamshell, and add 50.0 μL of cells from the P+ tube to the section marked “P+.” Close the lid. 
    4. Again, gently pump the pipette two or three times in the P+ tube to suspend the cells, and load 50 uL of the P+ cells. 
    5. Open the lid of the NA/amp plate, like a clamshell, and add 50.0 μL of cells from the P+ tube to the section marked “P+.” Close the lid. 
    6. Set the P-200 micropipette to 100 μL, gently pump the pipette two or three times in the P+ tube, and load 100 μL of the P+ cells. 
    7. Open the lid of the NA/amp/ind plate, like a clamshell, and add 100.0 μL of P+ cells to various areas across the surface—not just a single spot. Close the lid. 
16. Spread the cells from the P+ tube on your NA, NA/amp, and NA/amp/ind plates: 

    

    Figure 13.7. Spreading the E.coli onto the medium surface

    1. Open the package of sterile cell spreaders at the end closest to the spreader handles. Remove only one spreader and close the package to keep the others sterile. 
    2. Open the lid to the NA plate, like a clamshell, and evenly spread the cells on the P+ side of the plate (and only on this side) by gently moving the spreader across the agar surface. Close the lid. 
    3. Carefully spread the P+ cells on the NA/amp plate using the same spreader and technique. 
    4. Carefully spread the P+ cells on the NA/amp/ind plate using the same spreader. Then gently rotate the plate beneath the P+ spreader so that the cells can be spread over the entire surface of this plate. Close the lid. Hold the spreader by the handle and do not allow the bent end to touch any surface, as this will contaminate the spreader. Place the used spreader in the biohazard bag. 
17. Allow all three plates to sit right side up for five minutes. Using provided tape, tape all three plates together and label tape with your group number and class period. 
18. Place the plates in the 37°C incubator upside down to prevent condensation from dripping onto the cells.
19. Place all microfuge tubes, pipette tips, and cell spreaders in the biohazard bag. 
20. Incubate the plates for 24–36 hours at 37°C. 
21. Examine the plates and in your notebook record the amount of growth on each half. 

    

    Figure 13.8. E. coli transformed with a plasmid with a gene encoding fluorescent protein growing on NA+Amp+IND

22. Discard the Petri plates in the biohazard bag when directed to do so. 

Analysis

Table 13.9. Transformed E. coli producing colored molecules NA+AMP+IND

1. Look at the results of your transformation.  Fill out Table 13.2 with observations on whether you see growth or not on the different media.
2. Do your actual results match your predicted results?  If not, what differences do you see and what are some explanation for these differences?
3. How many colored colonies were present on your NA/AMP/IND plate? 

 

Table 13.2 Observations

Transformation of E. coli
Medium	No plasmid control	Treatment with plasmid
Nutrient Agar
Nutrient Agar + Ampicillin
Nutrient Agar + Ampicillin + Inducer