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

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