Protocol for preparing Wall-Associated Lysin (WAL)

Protocol for preparing Wall-Associated Lysin (WAL)

from PBCV-1 infected Chlorella-like algae NC64A cells
(Adapted from a thesis by Jane E. Kennedy, UNL, 1987)
D. Dunigan, University of Nebraska-Lincoln, 7/29/03

A. Cell growth and infection
1. Transfer NC64A cells into KBBM (BBM + 0.5 % sucrose + 0.01 % proteose peptone, pH ~6 + 50 mg/L tetracycline to prevent bacterial growth).
2. Grow 500 mL of cells to a density of ~ 1 x 109 cells/mL under fluorescent illumination with a 16 hr light/8 hr dark cycle at 25C on an orbital shaker (~95 rpm).
3. Collect cells by centrifugation at 4,100g for 5 min at 4C.
4. Re-suspend cells in 500 mL BBM + 8 mL of KBBM.
5. Infect cells with PBCV-1 at moi= 5 pfu/cell.
6. Incubate 10 - 12 hr at 25C with constant agitation.
B. Cell wall harvest and processing for WAL
1. After incubation, harvest cell walls by centrifuging 5 min at 4,800g (4C.)
2. Discard supernatant.
3. Wash pellet with 1 vol of BBM.
4. Resuspend pellet with 1/4 vol elution buffer (500 mM KCl, 20 mM Tris, 2 mM 2-mercaptoethanol, pH 7.5).
5. Incubate suspension at 37C for 1 hour with constant agitation.
6. Centrifuge 45min at 12,000g (4C) in SS-34 rotor, collect supernatant.
7. Dialyze supernatant 3x against (30 mM KCl, 20 mM Tris, pH 7.5, 2 mM 2-mercaptoethanol, 0.02 % NaN3) over 24 hrs at 4C.
8. Filter dialyzed supernatant through 0.2 mm filter, collect flow-through.
9. Concentrate flow-through with a Amicon UM10 or PM10 filter (10,000 MWCO), which reduces vol 10 - 20 fold.
10. Aliquot retentate into 1 mL fractions, store at 4C.
Bold's Basal Medium (BBM) Stock solutions:
1. 25.0 g NaNO3 per liter d-H2O
2. 2.5 g CaCl2-2H2O per liter d-H2O
3. 7.5 g MgSO4-7H2O per liter d-H2O
4. 7.5 g K2HPO4 per liter d-H2O
5. 17.5 g KH2PO4 per liter d-H2O 6. 2.5 g NaCl per liter d-H2O
7. 50.0 g Na2-EDTA, 31.0 g KOH per liter d-H2O
8. 4.98 g FeSO4-7H2O per liter acidified H2O (Acidified H2O is 999.0 mL d-H2O + 1.0 mL concentrated H2SO4)
9. 11.42 g H3BO3 per liter d-H2O
10. 8.82 g ZnSO4-7H2O, 1.44 g MnCl2-4H2O, 0.71 g MoO3, 1.57 g CuSO4-5H2O, and 0.49 g CoNO3-6 H2O per liter d-H2O
Autoclave each of these stock solutions.
BBM preparation*:
to 950 mL d-H2O add: 10.0 mL of stock solutions 1, 2, 3, 4, 5, and 6
1.0 mL of stock solutions 7, 8, and 9
2.0 mL of stock solution 10
Autoclave
*Tetracycline (filter sterilized, 10 mg/mL final concentration) is added after the media is autoclaved and cool.
KMMB preparation:
to 1 liter of BBM add:
5 g sucrose
0.1 g proteose peptone
Adjust pH to 6
Autoclave

Add filter sterilized tetracycline at 50 mg/L final concentration after medium is cooled.