This study investigates the effects of a mutant variant of DNA polymerase in genetically modified bacteria, focusing on the resulting mutations and their impact on beta-glucosidase expression. By analyzing bacterial cultures over generations, the research aims to quantify mutation frequency and enzyme activity.
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 37 degrees Celsius. The next morning, dilute the pre-culture 1 to 250 in three flasks containing 10 milliliters of fresh media. Add the inducers one millimolar each of arabinose, IPTG or arabinose and IPTG, and incubate the induced culture at 37 degrees Celsius for eight hours.
Prepare non-induced cultures in parallel. Collect one milliliter aliquots and dilute them one to 500 in new flasks containing 10 milliliters of fresh media, supplemented or not supplemented with inducers. Then incubate the mixture overnight at 37 degrees Celsius.
The next day, repeat the steps beginning with the dilution of the pre-culture and collect one milliliter aliquots. The aliquots are then placed in the freezer.
Next, determine the number of generations that have occurred in each culture. Transfer 100 microliters of appropriate serial dilutions of inoculum and culture on LB plates.
Incubate the plates overnight at 37 degrees Celsius. The following morning, count the colonies on the LB plates.
Calculate the log of the number of cells present in the inoculum or log I and in the culture at the end of growth or log C, and determine the number of generations.
Then centrifuge the culture at 5,000 gs for 20 minutes and resuspend the cells in one milliliter of 50 millimolar tris HCL, pH 7.6, 50 millimolar NaCl.
To permeabilize the cells, add two to three drops of chloroform and vortex for 20 seconds.
Finally, determine the glucosidase activity of each aliquot in a 96-well microplate.
Add 100 microliters of permeabilized cells and 100 microliters of using p-nitrophenyl-D-glucopyranoside substrate to each well, while avoiding the creation of air bubbles in the wells.
Read the absorbance at 420 nanometers using a microplate reader and filter.
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