Team:OLS Canmore Canada/Experiment

EXPERIMENT

Protocols Used Throughout the Duration of Project

Preparation of competent B. Subtilis SCK6 cells for immediate transformation:

  1. 2 days before transformation streak out a plate (1uL/mL final Erythromycin) of SCK 6 and grow at 37℃ overnight.
  2. Prepare seed culture: inoculate a single colony from the plate into 5 mL of LB-Erythromycin (1uL/mL) in a 50 mL Falcon tube. Incubate overnight at 37℃ at 200 rpm.
  3. Take the absorbency of the seed cultures using the spec. The OD600 should be approximately 1.0.
  4. Place 1000µL of overnight culture into a sterile Eppendorf tube for each transformation. Centrifuge and pour off the supernatant. Repeat 4 more times, to centrifuge the entire 5 mL of the overnight culture into 1 Eppendorf tube - pellet should be large. Do not discard the overnight falcon tube.
  5. Place 5 mL of Fresh LB broth (pre-warmed to 37℃) into the original overnight Falcon tube. Add 5 µL of erythromycin. Add 250 µL of 10% sterile xylose solution to the LB broth. Add 500 µL of this broth mixture to centrifuged cells in the Eppendorf tube (pipette up and down to resuspend cells). Transfer 500 µL of cells back into large Falcon tube. Incubate at 37℃ for 2 hours on the rotary shaking table.

Transformation of B. Subtilis

  1. Mix 2 µL of original stock, 2 µL of 1:10 diluted stock of the desired plasmid with 100 µL of competent cells in an Eppendorf tube.
  2. Incubate the cells at 37℃ at 200 rpm for 1.5 hours to complete the transformation.
  3. Spread transformed cells on LB plate with the appropriate antibiotic. Incubate the plates at room temperature overnight to select transformants.

Preparation of Lysis Buffers, Lysozyme

      Preparation of 0.5 L of 1 M Tris-HCl (pH 8) Stock:
    • Dissolve 60.5 g TRIS in 400 mL dH2O.
    • Adjust pH using 30 M HCl to pH 8 (tested with pH strips).
    • Make up volume to 500 mL with dH2O.
      Preparation of 500 mL 0.5 M EDTA Stock:
    • Dissolve 93.0 g EDTA (EDTA. Na22H2O) in 400 mL dH2O.
    • Add 10 g NaOH powder to adjust to pH 8.
    • Add dH2O to make up the volume to 500 mL.
      Preparing 500 mL TRIS + EDTA + SDS - Lysis Buffer:
    • Add 5 mL of 1 M Tris-HCl (pH 8) to 1 mL 0.5 M EDTA.
    • Add to 5 mL of 10% SDS solution (purchased) to 400 mL dH2O.
    • Mix, then add more dH2O to make up 500 mL.
      Preparing Lysozyme Solution (20 mg/mL):
    • Add 0.100 g of Lysozyme powder to 5 mL Tris buffer.
    • Dissolve.
    • (Makes 20 mg/mL STOCK)

Members of our team using the Lysis Protocol.

Lysis Protocol (E. Coli)

  1. Take 4000 uL of overnight cell culture cell, and transfer to a 2.0 mL Eppendorf tube (repeat as many times as needed for each culture, or multiple trials).
  2. Centrifuge cells at 4000 rpm to pellet cells at the bottom.
  3. Pour off supernatant, being careful not to disturb cells *Repeated 2x to get larger pellet.
  4. Re-suspended cells 400 uL of lysis buffer.
  5. Add 60 uL of 20 mg/mL lysozyme solution (made in tris buffer). Mix by gently tipping tubes back and forth.
  6. Incubate for 1 hour at 37℃, with gentle rocking

Spectrophotometer Readings - Cell Cultures

  • Place 900uL of sterile LB buffer in a cuvette. Use this to “blank” the spec.
  • Place 900uL of untransformed E. coli cell culture into a cuvette. Ensure the OD600 of the culture is between 0.5 and 1.0. Take absorbance readings at the following wavelengths: 557 nm, 562 nm, 567 nm, 572 nm, 577 nm, 582 nm, 587 nm, 592 nm, 597 nm, 60 nm, 607 nm, 612 nm, 617 nm.
  • Repeat step 2 a second time with the same cuvette, and average the two absorbance readings. Record this average.
  • Repeat steps 2 and 3 for the transformed cell cultures containing all 4 constructs.

Spectrophotometer Readings - Lysed Supernatants

Spectrophotometer Reading for Lysed Supernatants.

  1. Perform the above noted lysis protocol on untransformed E. coli, as well as transformants for all 4 new constructs.
  2. Spin down 1000uL of each lysis product in a centrifuge and save the supernatant.
  3. Transfer 900uL of lysis buffer to a cuvette. Use this buffer to “blank” the spectrophotometer.
  4. Transfer 900uL of supernatant from lysed, untransformed cells to a new cuvette. Take spec readings at the following wavelengths: 557 nm, 562 nm, 567 nm, 572 nm, 577nm, 582 nm, 587nm, 592 nm, 597 nm, 602 nm, 607 nm, 612 nm, 617 nm.
  5. Repeat step 4 a second time with the same cuvette, and average the two absorbance readings. Record this average.
  6. Repeat steps 4 and 5 for the lysed supernatants from all 4 constructs.

Spectrophotometer Readings - PET and non-PET Plastics:

Spec work.

  1. Cut several 5mm by 20mm pieces of clear, clean PET plastic (or another size that fits in your spec cuvette).
      2. Inside eppendorf tubes, soak one piece of PET plastic inside each of the following:
    • Distilled water
    • Sterile LB broth (negative control “C”)
    • Untransformed E. coli cell culture (positive control“A”)
    • Transformed E. coli with PET-ase construct (“E”)
    • Transformed E. coli with PET-ase mCherry construct (“F”)
    • Transformed E. coli with BslA construct (“G”)
    • Transformed E. coli with mCherry BslA construct (“H”)
    • Lysis buffer (negative control “C”)
    • Lysate from Untransformed E. coli culture (positive control “A”)
    • Lysate from Transformed E. coli with PETase construct (“E”)
    • Lysate from Transformed E. coli with PETase mCherry construct (“F”)
    • Lysate from Transformed E. coli with BslA construct (“G”)
    • Lysate from Transformed E. coli with mCherry BslA construct (“H”)
  2. Place the plastic in 900uL of dH2O in a cuvette, and use this to get a base reading of the absorbance of clear PET plastic at the following wavelengths: 557 nm, 562 nm, 567 nm, 572 nm, 577 nm, 582 nm, 587nm, 592 nm, 597 nm, 602 nm, 607 nm, 612 nm, 617 nm
  3. After 48 hours of soaking the plastics in their respective eppendorf tubes, each plastic piece was rinsed in dH2O for 5 seconds. This is to test the adherence of any proteins after a gentle rinse.
  4. Each plastic was then placed in 900uL of dH2O in a cuvette and scanned at 587nm to check absorbance.
  5. Steps 1-5 were repeated for a clear, non-PET plastic, and results also recorded.