8.4: Part I- Lysis of Bacterial Cells

Previously, bacteria were transformed with a recombinant plasmid capable of expressing gfp when cells were induced. The reason the cells can be induced to produce protein is that within the plasmid, in front of the gene for gfp, there is a special sequence of DNA that will respond if a chemical is placed in the media. This chemical is called an inducer and signals that the gene should be “turned on” and messenger RNA should be transcribed from the DNA instructions and the protein should be produced.  The plasmid also contains the selectable ampiclillin resistance gene to ensure that the cells growing in your culture are cells that contain your plasmid.

Prior to this lab period, cells were grown in the presence of ampicillin until late in log phase. The cells in culture divide and each cell contains many copies of the plasmid. At late log phase, the chemical inducer was added to the medium to turn on the gfp gene and the cells were allowed to continue to grow and produce gfp. 

First, you will collect your cells and break them open. This process is called cell lysis. After the cells are lysed, you will use column chromatography to purify gfp.

Materials

Reagents (changed slightly)

• Microfuge tube rack with the following tubes: 
  • LB/amp/ind culture of E. coli cells (EC) 

  • elution buffer (EB) 

  • lysis buffer (LyB) 

• Additional 1,000 μL (1 mL) of the LB/amp/ind culture of E. coli cells (obtain from your instructor in step 6)

Equipment and Supplies (changed slightly)

• P-200 micropipette 
• Tip box of disposable pipette tips 
• Permanent marker 
• Microcentrifuge (shared with class) 
• Vortex mixer (shared with class) 
• Long wave UV light
• Liquid waste container 
• Sharps container
• Biohazard bag for materials that come into contact with E. coli cells (shared with class)

Safety Reminder

Appropriate safety precautions should be used at all times. These will be reviewed by your instructor and can be found in the beginning of your laboratory manual which you should refer to before you begin this procedure. Aseptic technique is required when handling E.coli and materials that have come in contact with the bacterial culture. Remember that aseptic technique are the procedures used to protect your culture and samples from contamination but also protect you. 

1. Disinfect your work area and wash your hands before beginning an experiment.
2. Never touch anything that has come in contact with the E.coli. This includes pipettes, spreaders, and the interior of tubes. Pipet tips should never touch anything except the material to be transferred. Spreaders and pipettes should only be handled from the end that will not touch bacteria.
3. When handling petri dishes, only open the lid enough to work with the agar surface and then close the lid immediately. This will avoid contamination, such as fungal spores from the air, landing on your agar plate.
4. If something becomes accidentally contaminated, speak to your instructor to inquire if a replacement is appropriate and available. 
5. Avoid spills. If one occurs, notify your instructor immediately for help in cleaning it appropriately. 
6. Contaminated waste such as used microfuge tubes and cell spreaders will be placed in the biohazard bag. Pipet tips will be placed in a sharps container.
7. Only when directed to do so will you dispose of your used petri dishes in the biohazardous waste. 
8. Be sure to clean your work area and wash your hands before exiting the lab. 

Procedures

1. Check your rack to make sure that you have the reagents listed for Part I. 

2. Examine the EC tube with a long wave UV light and record its color in your notebook. 

3. Before you can lyse the cells, you will need to separate the cells from the growth medium. To do this, spin the EC tube in the microcentrifuge for five minutes.
4. Carefully remove the EC tube from the microcentrifuge to avoid disturbing the solid pellet that contains the bacterial cells. 

5. Set the P-200 micropipette to 200 μL, place a new tip on the micropipette, and carefully remove the supernatant (liquid) from the EC tube without disturbing the cell pellet. (You can dispense the supernatant into the liquid waste container.) 

6. Bring the EC tube to your teacher, who will add 1,000 μL (1 mL) of the LB/ amp/ind culture of E. coli to your EC tube. 

7. Repeat steps 3–5 (spin the tube for 5 minutes and remove the liquid). 
What color is the supernatant? The pellet? What are the contents of each? 

8. Use the micropipette to remove as much of the liquid as you can without touching the cell pellet.
9. Using the P-200 pipette, add 150 μL of elution buffer (EB) to the cell pellet in the EC tube. 

10. Close the cap of the EC tube tightly and resuspend the cells using a vortex mixer. If you do not have a vortex mixer you can repeatedly drag the tube vigorously across the surface of the microfuge tube rack. Examine the EC tube carefully. If there are visible clumps of cells, continue vortexing or dragging the tube to resuspend the cells.
11. Using the P-200 pipette, add 150 μL of Lysis buffer (LyB) to the EC tube. Close the cap of the EC tube tightly and mix the contents using the vortex mixer or by dragging the tube vigorously across the surface of the microfuge tube rack several times. 

12. Label the EC tube with your group number and class period and give it to your instructor. Your teacher will incubate the cells at room temperature overnight. 

13. Place all microfuge tubes and pipette tips in the biohazard bag.