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

Abstract

Source: Chen, Y., et al., Measurement of Specific Mycobacterial Mistranslation Rates with Gain-of-function Reporter Systems. J. Vis. Exp. (2019)

This video demonstrates a method to quantify mistranslation in Mycobacterium smegmatis using an inducible GFP and secreted mutant luciferase reporter system. It shows how luminescence resulting from mistranslation is measured and compared to GFP fluorescence to evaluate the impact of a test molecule on translational accuracy.

Protocol

1. Nanoluc luciferase (Nluc)/Green fluorescent protein (GFP) Reporter

NOTE: For a visual representation of this method, see Figure 1.

1. Inoculate the bacterial reporter strain from -80 °C stock with 2 mL of 7H9 medium. Shake at 37 °C for 1 to 2 days till OD₆₀₀ reaches the stationary phase (OD >3).
2. Subculture to 50 mL of 7H9 medium and grow till OD₆₀₀ reaches the late stationary phase (>4).
3. Before aliquoting the bacteria to a 96-well plate, add anhydrotetracycline (ATC) at a final concentration of 50 ng/mL and mix well. Induction of the bulk culture ensures that all wells contain the same amount of inducer and that the induction of the reporter is synchronized. Aliquot the bacteria to a clear, round-bottomed 96-well plate, with 100 μL of volume in each well.
4. To screen for small molecules affecting mistranslation rates, add the compound at the indicated concentration to select wells. For the purposes of this protocol, use kasugamycin (at doses indicated in Figure 2) as an illustration. Add different doses of kasugamycin to select wells (each experimental group should contain at least two biological replicates).
5. Shake and induce the samples at 37 °C for 16-20 h.
   NOTE: It is necessary to seal the plate with film. Also, all the edge wells need to be filled with at least 200 μL of sterile water to limit evaporation from the test wells.
6. Take 80 μL from each well, using a multichannel pipette, and transfer the samples to a black 96-well plate (which maximizes the fluorescence signal measurement). Measure the GFP signal by luminometer with 20 ms as integration time.
7. After measuring the GFP signal, centrifuge the plate at 3,220 x g for 10 min. Transfer 50 μL of the supernatant to a white-bottomed 96-well plate (which maximizes the luminescence signal measurement), add 50 μL of Nluc substrate to each well, mix them well, and measure the luminescence by luminometer with 1,000 ms as integration time.
8. Determine the Nluc/GFP ratio by dividing the corrected Nluc luminescence values by GFP fluorescence: this measure (in arbitrary units) is a relative measure of aspartate for asparagine mistranslation.

Results

Figure 1: Outline of measuring the mistranslation rate Nluc-GFP reporter. A fresh culture of...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Middlebrook 7H9	BD Difco	271310
Anhydrotetracycline	Cayman Chemical	10009542
Kasugamycin	Sigma	K4013
Nano-Glo luciferase assay	Promega	N1120
Fluoroskan Ascent FL luminometer	Thermo	/
Assay Plate, 96 Well White, Flat Bottom High Binding, No Lid	Costar	3922
Assay Plate, 96 Well Black, Flat Bottom High Biding, No Lid	Costar	3925
96 Well Cell Culture Cluster, Flat Bottom with Lid	Costar	3599
pUV-TetOR-Ag85ASec-Nluc-D140N	NA	NA	Episomal shuttle plasmid with a tetracyclin-inducible secretable version of mutated Nluc luciferase
pMC1S-GFP	NA	NA	Mycobacterial integrated (L5 site) vector for tetracycline-inducible expression of GFP