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Conjugation of E. coli to Pseudomonas

Using the oriT/ori1600 constructs submitted to the registry. A kanamycin resistance plasmid was transferred from E. coli to Pseudomonas putida and Pseudomonas fluorescens with the following protocol (see the Chassis section for more information). While this protocol is unusual for most biparental mating type experiments found in the literature, it underlies the speed and efficiency of the reaction that is taking place. Further experiments in the future will be used to quantitate the degree of plasmid transfer.

1. Set up a 5mL LB culture with appropriate amount of antibiotic marker for the plasmid to be transferred.
2. Inoculate the culture with one colony of the donor plasmid strain.
3. Subculture ~100 uL of the receiving plasmid into the solution and shake at 37°C overnight.
4. Determine the OD600 of each culture and plate 200 cells onto each resistance plate.
5. For E. coli to Pseudomonas transfer, plating can occur on McConkie agar and oxidase testing can be used to ensure transfer is happening.