Tri-parental mating

From ActinoBase

Tri-parental Mating

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

Tri-Parental Mating

Tri-parental mating is useful for conjugating large plasmids, such as PAC clones (see PAC cloning), or those containing a kanamycin resistane gene as a selectable marker into Streptomyces. In the standard ET Conjugation protocol a helper plasmid, pUZ8002, is used in the Escherichia coli strain ET12567 (E. coli ET12567) to transfer the desired vector to Streptomyces. This pUZ8002 helper plasmid contains a kanamycin resistance gene. This means that, if your desired plasmid contains a kanamycin resistance gene as marker (e.g. pRT802), it would be impossible to select for this vector when transforming E. coli ET12567 due to the kanamycin resistance gene already present on pUZ8002.

To circumvent this issue, tri-parental mating is used. This transforms E. coli ET12567 with your vector of choice (donated from a parental E. coli strain) and a different helper plasmid, pR9604, which contains an ampicillin/carbenicillin resistance gene as a selectable marker (donated from the parental strain E. coli TOP10 pR9604). The protocol then uses Amp/Carb to select for the helper plasmid, and kanamycin can be used to select for your desired vector. At the end an E. coli ET12567 strain is generated with an Carb/Amp selectable helper plasmid, and your kanamycin-marked vector of choice.

Protocol

Strains needed:

  1. E. coli TOP10 donor strain containing the self‐transmissible helper plasmid pR9604 (CarbR/AmpR).
  2. E. coli donor strain containing your vector of choice
  3. E. coli ET12567 recipient strain (ChlR)


Materials needed needed:

  1. Liquid LB medium
  2. Solid LB agar containing no antibiotics
  3. Solid LB agar containing chloramphenicol, carbenicillin/ampicillin and kanamycin
  4. Sterile loop/toothpicks


  • Sub‐culture overnight cultures of each strain into fresh LB and grow them to exponential phase. Note: the strains have different growth rates, so carry out a trial experiment under local conditions first. See the Antibiotic stocks and working concentrations page for recommended antibiotic concentrations.
  • Pellet cells via centrifugation, discard the supernatent and resuspend the pellet in fresh LB. Do this twice to remove residual antibiotics.
  • After a final spin, resuspend each pellet in 500μl of fresh LB.
  • Spot 10 to 20 μl of each strain onto the same location on the LB agar plates containing no antibiotics so that the three strains are mixed together. Wait for the spots to dry in and incubate at 37ºC overnight. (Note: Pilli are very fragile and sheared off in liquid culture so performing the triparentel mating on solid medium is recommended.)
  • The next day, prepare LB agar plates containing kanamycin, carbenicillin/ampicillin and chloramphenicol to select for E. coli 12567 derivatives containing both the helper plasmid, and your desired kanamycin-marked vector.
  • Using a sterile loop or toothpick, streak the overnight incubated spots onto the LB plates containing Kan, Amp/Carb and Chl for single colonies and incubate overnight at 37ºC.
  • After overnight incubation, inoculate single colonies into liquid LB medium broth containing Kan, Amp/Carb and Chl. If possible carry out PCR analysis to confirm the presence your desired vector.
  • This new E. coli ET12567 strain, containing both pR9604 and your desired kanamycin-marked vector can now be glycerol stocked, and used to conjugate your vector into Streptomyces via the standard ET Conjugation protocol.


Notes

  1. Ampicillin and Carbenicillin can be used intergangably.
  2. E. coli ET12567 contains a chloramphenicol resistance gene integrated into the genome.



Protocol adapted for ActinoBase by Samuel M.M. Prudence from the University of East Anglia.