* 3 ml pre-cultures of E. coli S17_1 and psychrophilic Pseudomonas sp. (or Pseudomonas putida instead or psychrophilic Pseudomonas) were used to inoculate an appropriate amount of LB to perform conjugation.
* E. coli was incubated for 4 h, and psychrophilic Pseudomonas sp. (or Pseudomonas putida) were incubated for 5 h.
* 2 ml E. coli S17_1 which contains target vector, and 2 ml psychrophilic Pseudomonas sp. (or Pseudomonas putida) were mixed by vortexing.
* The cells were centrifuged at 4000 g for 5 min.
* Supernatant was discarded.
* Pellet was dissolved in 100 ul LB and transferred to LB agar plates as one drop.
* The plates were incubated 1 day at 30ºC and 1 day at 20ºC.
* After conjugation, each drops were taken by sterile pipette tip from the agar plates and transferred into 1 ml of LB and diluted up to 10-4.
* 10-2 and 10-4 samples were plated out using LA containing kanamycin and chloramphenicol.
* LA plates with kanamycin were kept at 4ºC for the selection of psychrophilic Pseudomonas sp. from E. coli S17_1.
* LA plates with kanamycin and chloramphenicol were kept at 20ºC for the selection of psychrophilic Pseudomonas sp. (or Pseudomonas putida) from E. coli S17_1.
Reference:
Deniz Ünver(2012), Expression of Human Hormones in a Psychrophilic Pseudomonas species strains. Advance course report,NTNU, Norway