This study investigates the role of mistranslation in protein synthesis using a Mycobacterium smegmatis model. By measuring luciferase activity in response to test molecules, the research aims to elucidate mechanisms of protein expression regulation.
Take a culture of Mycobacterium smegmatis harboring inducible genes for GFP and a secretion signal-tagged mutant luciferase.
The mutant gene encodes an inactive luciferase, but natural mistranslation during protein synthesis produces an active enzyme.
GFP serves as an internal control to normalize luciferase expression.
Add the inducer to initiate gene expression, then aliquot the culture into a multi-well plate.
Introduce increasing concentrations of a test molecule to selected wells. Incubate.
Under control conditions, mistranslation results in active luciferase secretion.
If the test molecule inhibits mistranslation, active luciferase secretion decreases.
Transfer culture aliquots to a black plate and measure GFP fluorescence.
Centrifuge to pellet the cells.
Transfer the supernatants containing secreted luciferase and add the substrate, where active luciferase catalyzes its oxidation, producing luminescence.
Measure luminescence and calculate the Luciferase/GFP ratio; a lower ratio indicates reduced mistranslation due to the test molecule.
To begin, inoculate two milliliters of 7H9 medium with the bacterial reporter strain.
Shake at 37 degrees Celsius for one to two days or until the OD at 600 nanometers reaches the stationary phase.
Then, subculture in 50 milliliters of 7H9 medium and grow until the OD at 600 nanometers reaches the late stationary phase. Before aliquoting the bacteria to a 96-well plate, add ATC at a final concentration of 50 nanograms per milliliter and mix well.
Add 100 microliters per well to a clear, round-bottomed 96-well plate. Next, add different doses of Ksg to selected wells to screen its effects on the mistranslation rates. Add 200 microliters of sterile water to edge wells of the plate to limit the evaporation from the sample wells.
Seal the plate, shake, and induce the samples at 37 degrees Celsius for 16 to 20 hours. Use a multi-channel pipette to take 80 microliters from each well. Transfer the samples to a black-bottomed 96-well plate and measure the GFP signal by the luminometer.
After measuring the GFP signal, centrifuge the plate at 3,220 times g for 10 minutes. Then, transfer 50 microliters of the supernatant to a white-bottomed 96-well plate.
Then, add 50 microliters of Nluc substrate to each well, mix well, and measure the luminescence by the luminometer. Finally, determine the Nluc/GFP ratio by dividing the corrected Nluc luminescence value by GFP fluorescence.
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