Bacillus Genetic Stock Center Catalog of Strains Seventh Edition ...

GENERALIZED TRANSDUCTION WITH CP-54BERAdapted from: Lecadet, M.-M., M. O. Blondel, J. Ribier. 1980. J. Gen. Microbiol. 121:203-12.Assaying Phage CP54-Ber1. Prepare spores in one of the complete growth and sporulation media described in the previous pages(2×SG, NBYS, BP). Wash them in sterile distilled water, store them at 4°C for 3-4 days, and wash themagain. Heat to 65°C for 15 min. Cool. Store at 4°C.2. Mix 0.1 ml of a suitable phage dilution with 3 ml of soft PA agar (0.5% agar) and 10 8 spores.3. Pour the soft agar, spore, and phage mixture on PA agar (1.5% agar).4. Incubate at 35°C overnight. Plaques are turbid on B. thuringiensis berliner 1715, clear on B. cereus6A3 (NRRL-569).Propagating Phage CP54-Ber1. Mix 0.1 ml of phage dilution with 3 ml of soft NBY agar (0.5% agar) and 10 8 spores.2. Pour the soft agar, spore, and phage mixture on NBY agar (1.5% agar).3. Incubate at 35°C overnight.4. Flood plate with 3 ml NBY broth. Take up broth and soft agar layer in a 10 ml pipet.5. Pellet agar, cells by low speed centrifugation (5,000 × g)6. Filter the lysate through 0.8 micron Millipore filter.Transduction with Phage CP54-Ber1. Inoculate a single colony of the recipient strain into 10 ml NBYS and incubate at 30C with shaking for5-6 hours (OD 650 ≈ 3.5).2. Centrifuge at 5,000 × g for 15 min to pellet cells.3. Suspend cells in original volume of fresh broth. Cell titer will be ≈ 4×10 8 cfu/ml.4. Mix 0.8 ml of recipient culture with 0.1-0.2 ml of phage lysate, diluted to give a multiplicity ofinfection of ≥ 2.5. Incubate at 35°C for 30 min with mild shaking6. Plate 0.1 ml aliquots on selective media. Select for antibiotic resistance or base or vitaminprototrophy on HCO; select for amino acid prototrophy on MP18 with the appropriate amino acidomitted.31/Back to Table of Contents