This study outlines a method for transferring plasmids between E. coli cells using an F-pilus. The process involves mixing donor and recipient cells, allowing for the formation of transconjugants that carry antibiotic resistance genes.
Begin with donor E. coli cells carrying an F-plasmid in which a transfer gene is replaced by an antibiotic resistance gene.
These cells also contain a rescue plasmid that encodes the missing transfer gene.
Take equal volumes of recipient E. coli cells with a different antibiotic resistance gene in their DNA.
Mix both cultures in antibiotic-free nutrient media and incubate without shaking.
The donor bacteria’s rescue plasmid expresses the transfer gene, allowing the donor cell to build an F-pilus.
The pilus forms a bridge between two cells, allowing unidirectional plasmid transfer from the donor to the recipient, forming transconjugants.
Vortex the mixture to disrupt mating pairs and place it on ice to prevent further mating.
Spot the culture on agar plates containing transconjugant selectable antibiotics and incubate.
The transconjugants, carrying both antibiotic-resistance genes, survive and form colonies in the presence of both antibiotics, confirming successful F-plasmid transfer.
In duplicate, aliquot 100 microliters of donor cells and 100 microliters of recipient cells to 800 microliters of sterile LB media for one milliliter total volume. Allow the cells to mate at 37 degrees celsius for one hour without shaking. After an hour, vortex the cells for 30 seconds to disrupt the mating pairs.
Then, place the cells on ice for 10 minutes to prevent further mating.
Select for the transconjugant MC4100 cells that harbor the pOX38-Tc chloramphenicol knock-out by spotting ten microliter aliquots of each dilution on each half of the agar plates containing streptomycin and chloramphenicol. Repeat for both duplicate mixtures. Then, incubate the plates overnight at 37 degrees celsius.
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