SV1 transduction

Phage transduction using SV1

This protocol has been confirmed to work for the following organisms:

• Streptomyces venezuelae with ΦSV1

Protocol

Materials needed:

Instructions:

References

Protocol adapted for ActinoBase by Morgan Feeney from the University of Strathclyde.

To prepare SV1-lysate, 104 phage were added to 106 SV77 donor spores in 800 μl pre-warmed (45°C) soft nutrient agar (SNA) and poured onto Difco nutrient agar plates containing 0.5% glucose, 10 mM MgSO4 and 10 mM Ca(NO3)2. The plates were incubated at 30°C overnight, then flooded with 2.5 ml Difco nutrient broth (DNB) and incubated for 3-4 hr at room temperature. The phage-containing DNB soak-out was harvested and filtered through a 0.45 μm filter to eliminate bacterial contamination. For transduction of the bldD::apr allele, 109 phage particles harvested from the bldD::apr mutant strain SV77, were mixed with 107-108 WT spores and incubated overnight on MYM agar at room temperature before overlaying with apramycin for selection. Plates spread with the recipient strain or the phage alone were used as controls. Transduction of the bldD::apr allele was confirmed by PCR using test primers listed in Table S4, and the strain was named SV74.