JoVE Encyclopedia of Experiments
Microbiology
0 views • 4:00 min • October 30th, 2025
Take a culture of genetically modified bacteria.
The bacteria are induced to express a mutant variant of DNA polymerase with defective proofreading activity, which increases the chances of replication error.
These errors can lead to spontaneous mutations in the regulatory region, which derepress the beta-glucosidase gene and trigger the expression of the beta-glucosidase enzyme.
Collect samples at regular time intervals and evaluate the number of bacterial generations in each culture. Centrifuge the samples and discard the supernatant.
Resuspend the pellet in a buffer.
Add a permeabilizing solution and mix to permeabilize the bacterial membrane.
Transfer the permeabilized bacterial samples to a multi-well plate.
Add a substrate that enters the bacteria and reacts with the beta-glucosidase enzyme, producing a colored product.
Measure the color intensity to determine the beta-glucosidase activity.
Analyze the beta-glucosidase activity in the bacterial cultures to determine mutation frequency across generations.
Transfer a single colony of E. coli TOP10 containing the pBAD-ε and the pGOOD1-εD12A11 vectors to one milliliter of LB media treated with antibiotics.
Incubate the culture overnight at 3
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