JoVE Encyclopedia of Experiments
Microbiology
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Take a culture of genetically modified spirochete bacteria. These bacteria carry the gentamicin-resistance gene.
Add a bacteriophage suspension to the bacterial culture.
The phages carry plasmid DNA encoding a kanamycin-resistance gene.
Incubate the mixture.
The bacteriophage attaches to the bacterial surface and injects the DNA into the cell.
The plasmid replicates independently and is passed on when the bacteria divide.
Mix the incubated culture with a molten agarose medium supplemented with gentamicin and kanamycin antibiotics.
Pour the mixture into a plate. Allow it to solidify and then incubate.
Under double-antibiotic selection, only cells carrying both resistance markers grow and form colonies, while the remaining cells die.
Pick a colony and inoculate it in a medium containing both antibiotics.
Incubate to facilitate the growth of bacteria with the transduced plasmid for further downstream analysis.
Decant the supernatant and resuspend the pellet in 14.5 milliliters of fresh BSK. Add less than or equal to 500 microliters of PEG-precipitated phage sample to the culture of the recipient clone.
After mixing well, incubate at 33 degrees Celsius for 72 to 96 hours. Perform the selection of transductants by solid-phase plating after mixing with PEG-precipitated phage as described in the manuscript. Depending on the background of the recipient clone, check that the colonies appear within the agarose on the selection plates after 10 to 21 days of incubation.
Pick at least 5 to 10 colonies that grow on the plate in the presence of both antibiotics using sterilized cotton-plugged 5.75-inches borosilicate pipette and inoculate them into 1.5 milliliters of BSK with the appropriate antibiotic.