Griffitts:Triparental Mating

Getting the strains growing

• On the day prior to setting up the mating, patch out the three strains involved (from single colonies) onto selective LB plates:
  • A recipient S. meliloti strain (this should be Sm-resistant)
  • The E. coli helper strain (usually MT616 or B001, on LB-Cm)
  • The E. coli donor (usually a plasmid-bearing DH5α strain)

Broth-based method

Mating Mix

• With a wooden applicator stick, scrape each strain and separately resuspend as dense suspensions in 200 μL liquid LB
• Combine the three strains as follows:
  • 80 μL of the S. meliloti recipient strain
  • 20 μL of the helper strain
  • 20 μL of the donor strain
• Transfer 100 μL of this mixture onto a plain LB plate and spread
• Incubate at 30°C for 12–24 hours

Recovery

• To the lawn of cells add 3 mL of liquid LB-10% glycerol
• Use a sterile spreader to resuspend the cells into the LB-10% glycerol
• Distribute this “mating mix” into microcentrifuge tubes
• Plate out on selective medium
  

Note: The mating mixes can be frozen away at this point

Selection

• Dilute the concentrated mating mix (above) 10X, 100X, and 1000X into LB broth
• Plate 100 μL of each onto selective LB plates (e.g. LB-Sm-Nm)
• Incubate at 30°C for 24–48 hours
  

Note: After a couple of days, you should see transconjugants growing, often with an accompanying background haze of cells (satellite colonies). Transconjugants must be restreaked to singles to get rid of these would-be hijackers.

Agar-based method

Mating "X"

• Using toothpicks, swipe each of the two E. coli strains onto a plain LB plate, forming an “X”
• On the middle of the “X” smear a large amount of the recipient Sinorhizobium strain
• Incubate at 30°C for 24 hours

The steps of a triparental mating.

Recovery and Selection

• Using a toothpick pick up a glob of bacteria from the zone where the mating occurred
• Streak onto the appropriate selective medium
• Incubate at 30°C for 24–48 hours
• Recover single colonies onto the appropriate selective medium