Team:British Columbia/Protocols/Bacteria

Competent Cell Preparation (Small Scale)

Protocol generously donated by Jeanette (Beatty lab manager).

Supplies needed:

• DH5(alpha)
• LB broth--about 50mL per culture (each culture makes about 25-30 * 100 uL aliquots); about 5 mL for the initial overnight culture (inoculates multiple cultures)
• 0.1M CaCl2 (filter sterilized and chilled)--about 40-50 mL per culture
• Ice water bath
• 60% glycerol--about 1 mL per culture
• Spectrophotometer
• 100-250 mL flasks, centrifuge tubes (50 mL Falcon or equivalent), microcentrifuge tubes

Note: When working with cultures (that you will use later), always work aseptically. That is, work near a flame and be sure to turn off the flame at the end and follow aseptic procedures.

Steps: 1. Inoculate 5mL overnight culture (see overnight culture protocol) and grow at 30°C. You can use a rotating incubator. Approximately 30°C (can be a little higher or lower) seems to be fine. Otherwise, shaking speed ~200 rpm.

Note:A possible source of DH5(alpha) are frozen aliquots of DH5(alpha) competent cells. You can use the whole aliquot.

2. Dilute 0.5mL overnight culture into 50mL LB and incubate at 30°C, shaking vigorously. Label with name, date, strain, flask # (when making more than 1 culture), etc. Shaking speed ~200 rpm.

• Note: You can inoculate and use several 50 mL LB cultures at the same time. A 125 mL or 250 mL Erlenmeyer flask works well as a culturing container. Make sure to not rip or contaminate the aluminum foil cover. When you’re done using the flasks, rinse with 10% bleach, then with water and put into dirty basket (ask Tony Lam for help if needed).

3. Harvest at Abs600nm = 0.401. Higher than this seems to work well anyway. Pipet the cultures into 50 mL Falcon tubes (don’t overfill).

Using a spectrophotometer: Take 1 mL from the culture and 1 mL from a LB stock and put into cuvettes. Turn on the spectrophotometer (on Antonio’s bench; make sure to ask if he needs to use it and ask him if you need help). Wipe the cuvette with Kimwipe (to clean the part where light passes through). Press the OD600 button. Put the 1 mL LB cuvette into the slot and press the Blank button. Replace the 1 mL LB cuvette with 1 mL culture cuvette and press sample. Rinse the cuvettes twice with 70% ethanol and twice with sdH20. Turn off spectrophotometer.

Note: You don’t have to work aseptically when using the spectrophotometer. However, as always, transferring cultures that you want to use later on means you should work aseptically.

4. Centrifuge at 1600g for 7 minutes (4°C).

5. Pour out the supernatant aseptically (also flame mouth and cap before and after). Wash gently in ~20mL COLD filter sterilized 0.1M CaCl2 (i.e. put the tube on ice or put in cooler). Swirl the tube using your wrist until the pellet disappears.

6. Filter sterilization: Use a syringe to suck up some CaCl2 (from a beaker probably). Attach a 0.4 micron filter unit to the tip. Press the plunger. Whether any part above the filter is sterile is unimportant; anything that passes through the filter is sterile. Make sure the filtrate enters a sterile container (e.g. Falcon tube). You can re-use the filter: remove the filter, use the syringe to suck up more CaCl2 and repeat. The original packaging material can hold the filter while you do this. Make sure the first few drops of filtrate does not go into the container with your previously sterilized CaCl2. You can drop it back into the unsterilized CaCl2. Label finished product.

7. Spin down gently: 1100g, 5 minutes, 4°C. See 4 about using the centrifuge.

8. Pour out the supernatant aseptically (flame mouth and cap before and after too). Resuspend in 12.5mL cold 0.1M CaCl2. Wrist.

9. Keep on ice 40 minutes. 10. Spin down: 1100g, 5 minutes, 4°C. See 4 about using the centrifuge.

11. Pour out the supernatant asepticall (flame mouth and cap before and after too). Resuspend in 2-2.5mL cold 0.1M CaCl2. Wrist.

12. Store over night at 4°C

13. Add glycerol to 15% and divide into 100uL aliquots, store at -80°C in Lagally Lab freezer; label with name, date, strain, flask where it came from, etc. Adding about 1 mL 60% glycerol to 2.5 mL of the final resuspended cells yields a ~15% glycerol solution.

14. Throw away your used tubes, pipets, filters, syringes, etc.

Bacterial Transformation

1. Thaw 50uL competent cells on ice for 15min
2. Add 1-2uL of your plasmid or ligation mix
3. Keep on ice for 45min
4. Heat shock at 42 degrees Celsius for 45 seconds
5. Keep on ice for 2min
6. Add 250uL of LB or SOC media
7. Incubate (while rotating/shaking) for 1 hour at 37 degrees Celsius
8. Spread 50uL and 200uL on selective media plates
9. Incubate plates at 37 degrees Celsius overnight and you should see colonies!

Protein expression of C41 DE3 cells with a lac promotor

Note: This protocol was done specifically for a lac promotor, the limonene synthase composite part

1. Pick a colony, grow in 50 ml LB in a 250 ml erlenmeyer flask with the appropriate antibiotics, overnight
2. Take 1 ml of the solution and put it into a 500 ml terrific broth solution in a 2 litre flask with appropriate antibiotics
3. Grow the cells until = 0.8
4. Add IPTG to a final concentration of 1 mM
5. Express cells at 20 degrees (or 16 degrees) at 205 rpm overnight
6. Spin down cells
7. Store pellet in -80 for use