SV1 transduction

Phage transduction using SV1

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

• Streptomyces venezuelae with ΦSV1

Phage transduction

SV1 is the best-established transducing phage for use in Streptomyces and is specific for use with S. venezuelae.^[1]

Phage transduction is useful for moving alleles (usually marked with an antibiotic resistance marker or other selectable marker) into another genetic background. For example, after deleting a gene using PCR targeting (a.k.a. "Redirect") it is often best to move this allele into a clean wild-type background (for an example, see [2]).

Protocol

Materials needed:

1. Soft nutrient agar (SNA)
2. DNA agar plates
3. DNB
4. MYM or another appropriate medium for growth of your strains
5. Antibiotic needed to select for the allele being transduced
6. SV1 phage (see High titre preparation of phage for how to prepare this)
7. Spore stocks of the appropriate S. venezuelae strains (e.g., the donor stain containing the allele you wish to transduce, and the recipient strain that you wish to move this allele into)
8. Sterile glucose (i.e. 20% w/v glucose)
9. Sterile MgSO₄ stock solution
10. Sterile Ca(NO₃)₂ stock solution
11. 0.45 μm filters
12. Spreaders, pipettes, toothpicks, Eppendorf tubes, etc.

Instructions:

1. You should titer your spore stock and phage before beginning.
2. First, prepare a lysate of the SV1 phage on your donor strain (the strain containing the allele you wish to transduce):
   1. Prepare DNA plates containing 0.5% glucose, 10 mM MgSO₄ and 10 mM Ca(NO₃)₂ and molten SNA (not too hot - should be ~45°C)
   2. Add 10⁴ of SV1 phage to 10⁶ donor spores in 800 μl pre-warmed (45°C) soft nutrient agar (SNA)
   3. Pour this mixture onto your DNA/glucose/MgSO₄/Ca(NO₃)₂ plates and let the molten agar solidify.
   4. Incubate the plates at 30°C overnight.
   5. Harvest the phage by flooding the plate with 2.5 ml DNB and incubate for 3-4 hr at room temperature.
   6. Collect the phage-containing DNB soak-out and filter through a 0.45 μm filter to eliminate bacterial contamination.

1. To then transduce your marked allele into the recipient strain:
   1. First, titer the new phage lysate and the spore stock of the recipient strain
   2. Mix 10⁹ phage with 10⁷-10⁸ spores of the recipient strain
      1. Controls here should be phage alone, and recipient strain alone.
   3. Spread on MYM agar (or another appropriate medium depending on your strain's growth requirements).
   4. Incubate overnight at room temperature before overlaying with the appropriate antibiotic that will select for the mutant allele you are transducing.
   5. Incubate plates at 30°C until transductants appear. These should be restreaked several times on appropriate medium (e.g. MYM) to confirm their phenotype (antibiotic resistance) and to purify away any remaining phage particles.

Notes

1. You should confirm the genotype of your transductants using an appropriate method (PCR, Southern blotting, whole genome sequencing.)

References

[1] Stuttard, C. “Transduction of auxotrophic markers in a chloramphenicol-producing strain of Streptomyces.” Journal of general microbiology vol. 110,2 (1979): 479-82. doi:10.1099/00221287-110-2-479

[2] Tschowri, Natalia et al. “Tetrameric c-di-GMP mediates effective transcription factor dimerization to control Streptomyces development.” Cell vol. 158,5 (2014): 1136-1147. doi:10.1016/j.cell.2014.07.022

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