Measuring Mycobacterium smegmatis Mistranslation Rates Using a GFP/Luciferase Reporter System

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