Protein synthesis is error-prone. However, errors may be adaptive under physiological stress. We've previously shown that specific translation errors in mycobacterium are contributed to antibiotic tolerance.
Being able to measure specific error rates allows us to target this bacteria-adaptive mechanism. The main advantages of these techniques are the gain-of-function reporters can be exquisitely sensitive in measuring small error rates that are not easy to detect by mass spectrometry. The Nluc/GFP assay allows the medium-throughput screening since it avoids excessive handling and lysis of the bacteria.
Demonstrating the procedure will be PhD student Yue-Meng Chen. Today we will demonstrate the procedure of using these two reporters through this video. To begin, inoculate two milliliters of 7H9 medium with wild-type and mutated mycobacterial reporter strains.
Shake at 37 degrees Celsius for one to two days or until the OD at 600 nanometers reaches the stationary phase. Aliquot and dilute to obtain an OD at 600 nanometers around 0.5 to 1. Then, add ATC to a final concentration of 50 nanograms per milliliter.
Immediately add different doses of ksg to the culture according to the manuscript to measure its effects on mistranslation rates. Incubate at 37 degrees Celsius with shaking for four to six hours. Next, transfer the bacterial cultures to a two milliliter tube.
And centrifuge at 3, 220 times g at room temperature for five minutes to pellet down the bacteria. After the centrifugation step, discard the supernatant and disrupt the bacteria by adding 40 microliters of 1x passive lysis buffer. Transfer the resuspended bacterial lysate to a white 96-well plate, one well per sample, and shake at room temperature for 20 minutes.
Add 80 microliters of firefly substrate to each well. Shake for 15 seconds and measure the luminescence by a luminometer. Then, add 80 microliters of Renilla substrate to each well.
Shake for 15 seconds and measure the luminescence by the luminometer. Use the corrected values to calculate the mistranslation rates of each condition. 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. In this work, the Renilla firefly reporter system was used to measure the mistranslation rate in the presence of kasugamycin in wild-type and in a strain of S.Smegmatis which ksgA was deleted.
Results showed a clear dose-dependent manner of ksg decreasing mistranslation rate while in the ksgA-deletion strain, ksg was less potent and the baseline of mistranslation rate was higher due to the resistance to modulation by kasugamycin. Kasugamycin action on mistranslation was also measured using the Nluc/GFP reporter. Both Renilla firefly and Nluc/GFP reporter involve measuring luciferase activity.
Small pipetting errors could cause big fluctuations in readouts. For starters, we suggest increasing the number of each replicates to minimize the chance of getting unreliable readouts.
