Team:Austin UTexas/Protocols

Protocols

Materials Needed

  • MRS liquid media
  • L. plantarum cells
  • Glass culture tube
  • CO2 incubator
  • MRS liquid media, containing 0.5 M sucrose
  • Spectrophotometer
  • Centrifuge
  • Vortex
  • 50 mL Falcon conical tubes (2)
  • 30% PEG 8000

Preparation of Recipient Cell Stocks

  1. Inoculate 10-25 mL of -80 &#2103 L. Plantarum stocks in MRS broth at 37 &#2103 CO2 incubator, overnight without shaking.
  2. Subculture by incubating culture in a 200 mL of prewarmed MRS broth. Add culture until OD600 is 0.1.
  3. Subcultures should be grown to an OD600 of 0.6 (4-8 hours).
  4. For glycine supplementation, the MRS broth should contain 0.5%-1.0% glycine.
  5. For NaCl supplementation, inoculate subcultures with 0.9M of NaCl.
  6. Harvest cells from the subculture by centrifugation at 4 &#2103 for 10 min. at 7000 rpm after inoculation.
  7. Rinse the cells in 200 mL of sterile, distilled water (~4 &#2103) and centrifuge again.
  8. Resuspend the pellet in 2-3 mL of cold, sterile, distilled water and aliquot 1 mL volumes to 1.5 mL microcentrifuge tubes.
  9. Centrifuge aliquots at 15,000 rpm for ~90 seconds and remove the supernatant.
  10. Rinse the cells twice with 1 mL of cold, distilled, sterile water. Then rinse with 1 mL of cold, sterile 30% PEG-8000 solution. Remove the supernatant.
  11. Suspend cells in 0.5-0.6 mL of 30% PEG for storage at -80 &#2103. These should retain viability for up to 2 years.

Treatment of Cells Prior to Electroporation

  • MRS broth was prepared using 55g of Difco MRS broth powder in 1L of distilled water. The solution was then autoclaved and stored in a 4 degree refrigerator along with the Difco MRS powder.
  • MRS agar was prepared by mixing 55g of Difco MRS broth powder and 15g of agar flakes in 1 L of water. The solution was then autoclaved and stored in a 4 degree refrigerator along with the Difco MRS powder.

Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat.