Team:Uppsala/Experiments


Experimental Procedures

Microfluidics

Transformation on chip:

Preparing for cell transformation

  1. Set up chip by connecting 0.583 mm tubes insulated with paper towels to the heat channels. Use bent metal connectors. Connect tubes to two peristaltic pumps; one for each heating channel. Place a syringe needle in the inlet of the transformation channel and a tube in its outlet. (See image of setup)
  2. Heat the chip by running water at 65°C and 300 mL/h through the heating channels.
  3. Take out 50 µL of heat shock competent cells from -80°C freezer and thaw them on ice for 5 min.
  4. Take 10 µL of competent cells into a separate tube for negative control.
  5. Add 0.8µL of DNA (at a concentration of approximately 70 ng/µL ) to the remaining 40 µL of cells. Incubate cell and DNA suspension for 20 min on ice.
Running the transformation chips
  1. Flush the cell channel with 2 mL of 75% ethanol with a syringe. Dry the cell channel with air. This cleaning step should be repeated after each transformation to avoid contamination.
  2. For each transformation: pipette 6 µL of cell and DNA suspension into the syringe connector on the chip.
  3. Push the suspension into the transformation channel by pushing air in with a syringe.
  4. Fill the transformation channel and heat shock the suspension for 45 sec.
  5. Push the cell suspension through and collect in an Eppendorf tube filled with 100 µL SOB.
  6. Place the tube on ice immediately.
  7. Incubate for 1.5 h at 37°C and then spread on an agar plate with the appropriate antibiotic.

General

Most of the general lab work, such as assemblies, were performed following the protocols in Synthetic Biology: A lab manual.

Liljeruhm, J. Gullberg, E. Forster, AC. 2014. Synthetic Biology: A lab manual. World Scientific Publishing Co. Singapore

For the IMAC purification we used the BioRAD IMAC Instruction Manual as a source for most of our work.

Competent cells

Materials

  • LB medium
  • Overnight culture
  • TFBI buffer
  • TFBII buffer
Volume of overday culture: 200 ml


Procedure

  1. Dilute the overnight culture 2 ml into 200 ml LB medium.
  2. Grow culture at 37ºC with shaking to an OD600 = 0.3 - 0.4.
  3. Transfer culture to 4 pre-cooled 50 ml sterile falcon tubes. Put on ice for 5 minutes.
  4. Centrifuge at 3000 rpm for ten minutes at +4°C.
  5. Discard the supernatant. Then resuspend the pellets in circa 5 ml TFBI. Then pour all mixtures together in one of the falcon tubes, and fill up to 40 ml with TFBI. Put on ice for 5 minutes.
  6. Centrifuge as above. (3000 rpm for 10 minutes at 4°C).
  7. Discard the supernatant. Then resuspend the pellet in 40 ml TFBI. Then put on ice for 5 minutes.
  8. Centrifuge as above. (3000 rpm for 10 minutes at 4°C).
  9. Discard the supernatant and resuspend in 6 ml TFBII. Then put on ice for 15 minutes.
  10. Aliquot 50 µl into Eppendorf tubes.
  11. Snap freeze with liquid nitrogen. Store in -70°C.

TFBI buffer

Materials

  • KC2H3O2 (potassium acetate)
  • RbCl CaCl·2H2O
  • MnCl2·4H2O
  • 15% v/v glycerol
Final volume: 1000 ml


Procedure

  1. Add the following to a glass flask:
    • 2.945 g KC2H3O2
    • 12.09 g RbCl
    • 1.47 g CaCl2·2H2O
    • 9.895 g MnCl2·4H2O
    • 150 ml 15% glycerol
  2. Add 850 ml of autoclaved ddH2O.
  3. Sterile filter and store at +4°C.
  4. Adjust to pH 5.8 with concentrated CH3COOH.

TFBII buffer

Materials

  • MOPS
  • RbCl
  • CaCl2 *2H20
  • 20% glycerol
Final volume: 1000 ml


Procedure

  1. Add the following to a sterile, autoclaved glass flask:
    • 2.095 g MOPS
    • 1.21 g RbCl
    • 11.025 g CaCl2 *2H20
    • 150 ml 20% glycerol
  2. Fill glass flask with double distilled, autoclaved water to receive wanted volume.
  3. Adjust pH to 6.6 with 5 M NaOH
  4. Sterile filter and store at +4°C.