Team:Austin UTexas/Protocols

Protocols


Supplies

  • 10X T4 DNA ligase buffer
  • T7 DNA ligase
  • Restriction endonuclease BsaI or BsmBI
  • Water

Setting up the Golden Gate assembly reaction

  1. In a 0.5 mL microcentrifuge tube, mix 20 fmol (1 nM final concentration) of the backbone and 40 fmol of each DNA insert together. The insert to backbone ratio should be 2:1. For the assembly of a transcriptional unit use a 3:1 insert to backbone ratio. To calculate the precise amount of DNA to add, use this calculator provided by the Barrick lab.
  2. Add water to a final volume of 16 µL.
  3. Add 2 µL of 10X T4 DNA ligase buffer. Mix by vortexing.
  4. Add 1 µL of BsaI or BsmBI and 1 µL of T7 DNA ligase. Mix by pipetting gently.

Thermocycler conditions for BsmBI assembly reactions

  1. 42° C for 5 minutes.
  2. 16° C for 5 minutes.
  3. Repeat steps 2 & 3 for 30x.
  4. 55° C for 10 minutes.
  5. Use 2 uL of this assembly reaction for electroporation.

Thermocycler conditions for BsaI assembly reactions

  1. 37° C for 5 minutes.
  2. 16° C for 5 minutes.
  3. Repeat steps 2 & 3 for 30x.
  4. 37° C for 10 minutes.
  5. Use 2 uL of this assembly reaction for electroporation.

  • The above Golden Gate assembly reaction protocol and thermocycler conditions used were directly from the Barrick lab protocols here.


Supplies

  • 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° C L. Plantarum stocks in MRS broth at 37° C 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° C for 10 min. at 7000 rpm after inoculation.
  7. Rinse the cells in 200 mL of sterile, distilled water 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° C. These should retain viability for up to 2 years.

Treatment of Cells Prior to Electroporation

  1. Thaw on Ice.
  2. Suspend cells in 900 μL of cold, sterile, distilled water for 30 min.
  3. Treat with 100mM lithium acetate and 10mM DTT solution for 30min.
  4. After pretreatment, the cells were pelleted for 2-3 minutes in a microcentrifuge, washed once in 1 mL of cold, sterile, 30% PEG solution and suspended in 0.5-0.6 mL cold, sterile PEG solution for electroporation.

Electroporation

  1. Isolate intended vector (Lactobacillus plantarum experiment as of 09/06/17 has been using pMSP3535).
  2. 200 ng/transformation of plasmid DNA is mixed with 100 μL of cell suspension and transferred to a prechilled electroporation cuvette with a 0.2 cm gap.
  3. Pulse at 12.5 kV (From literature: Bio-rad gene pulser with capacitance setting at 25 μF and resistances of 200 or 400 Ω and voltage settings of 1500, 2000 or 2500 V (7.5, 10 or 12.5 kV cm-1).
  4. Quickly recover cells by adding 900 μL of recovery medium (0.5 M sucrose in MRS broth) to the cuvette then transfer the cells to a sterile microcentrifuge tube for incubation ~4 hours at 37° C in a CO2 incubator without shaking. (*Save recovery and plate again if no growth)
  5. Dilute cells in recovery medium and plate on MRS agar along with the proper concentration of any antibiotics that the plasmid of choice has resistancy (pMSP3535 has resistance to erythromycin).
  6. Incubate plates for 2 days, then count the colonies present. Allow an additional 3-4 days after initial count to ensure all were noted.
  7. Cells stored in glycerol (-80° C) after electroporation incubation can be expected to yield similar results to the cells that were plated directly after the incubation.

References

  • Welker, D. L.; Hughes, J. E.; Steele, J. L.; Broadbent, J. R. High Efficiency Electrotransformation of Lactobacillus casei. FEMS Microbiology Letters 2014, 362 (2), 1.
  • Spath K, Heinl S, Grabherr R. Direct Cloning in Lactobacillus Plantarum: non-methylated plasmid DNA enhances transformation efficiency and makes shuttle vectors obsolete. Microb Cell Fact 2012, 11 (141).

Lactobacillus plantarum is a gram-positive lactic acid producing bacteria, so it requires a different growth media than we typically use in our lab. We have exclusively grown ourbacteria on MRS media. Further, we grew Lactobacillus plantarum in a CO2 incubator as referenced in most literature we studied, for example Welker et. al. (2014).


  • 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.

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