The protocol allows mutational estimation in microbes. It can demonstrate how an organisms environmental context effects probability of spontaneous mutation. The main advantage of this protocol is that it's cheap and efficient.
Many estimates of mutation rate can be made in parallel. Mutations that this protocol measures could follow resistance to antibiotics. So this protocol can be used to study one of the world's biggest challenge, antimicrobial resistance.
This method can provide insight into how cells ecological context can effect the evolution of antimicrobial resistance. This protocol estimates mutational rates in the monoculture of laboratory strain, but we have applied it to clinical strains and co-culture of two strains to distinguish bioneutral molecule. The most hazardous reactions used in this protocol are antibiotic rifampicin and methanol.
So make sure to use protective gloves and goggles when rifampicin is dissolved in methanol. To begin this procedure inoculate three mL of liquid lysogeny broth with a scrape of ice from the E.coli K12 glycerol stock. Shake the LB culture at 120 rpm and at 37 degrees celsius for approximately seven hours.
After this, dilute the culture 2000-fold with the saline solution. Add 10 mL of liquid Davis minimal medium to each tube with each containing a different concentration of glucose as shown here. Add 100 microliters of the diluted culture to three 50 mL screen cap conical bottom polymer tubes.
Prepare 22 mL of five different solutions of liquid Davis minimal medium with glucose, each in its own 50 mL tube as outline in the text protocol. To prepare inocula for the environments, first measure the optical density of the overnight cultures at 600 nanometers. Dilute the cultures with saline solution and add between 2200 and 110000 cells to each environment.
This will ensure that the inoculum for one mL of the environment contains between 1000 and 5000 cells. Next, create a random layout of parallel cultures for 196 deep well plate. Transfer one mL of the inoculated media into each well of the plate according to the layout.
Then, fix the lid of the plate with tape and weigh the entire plate with the lid and tape. Shake the plate at 250 rpm and at 37 degrees Celsius for 24 hours. After this, place two L of distilled water in the incubator to stabilize the amount of evaporation among the experimental sets.
Determine the inoculum size by plating 10 microliters of each of the inoculated media on a non-selective TA agar plate. Use a sterile L-shaped spreader until the agar surface is dry. Then, prepare selective TA agar containing rifampicin in six well plates.
Pipette five mL of the selective TA agar into each well of the six well plates. Count the colony forming units on the non-selective agar plates, and determine the size inocula. After 24 hours of incubation, weigh the entire deep well plate to determine the amount of evaporation, which is likely around 10%Transfer three randomly chosen cultures per assay into labeled microcentrifuge tubes.
Plate the remaining 81 parallel cultures from the deep well plate onto the selective TA agar containing rifampicin. Next, remove the lids from the selective agar plates and leave them uncovered in sterile conditions to dry out all of the liquid on the surface of the agar. Determine the number of colony forming units by diluting the cultures from the microcentrifuge tubes using five 10-fold dilution steps by mixing and vortexing 900 microliters of saline solution with 100 microliters of the culture per dilution step.
Plate 40 microliters of the final dilution on the non-selective TA agar and place the lid on the plates. Incubate overnight at 37 degrees Celsius. Once all of the wells on a six well plate are free of the culture liquid, place the lid back on.
Then incubate the plates with the lid on at 37 degrees Celsius for 44-48 hours. On day four, count the colony forming units on the non-selective agar plates. On day five, count the number of colonies that are resistant to the antibiotic on the selective TA agar plates.
Record the distribution among parallel cultures of the observed number of mutants for a particular assay. Open the appropriate software on the computer. In the hypothesis testing tab, leave the values at their defaults.
Click browse and select the text file with the distribution of observed mutants. After uploading the file, click on performed test. On the right side, under the result of the test, under the One Sample ML-test, find the mutation number.
This is m, the expected number of mutational events. Then, estimate the mutation rate of particular genotype in a particular environment as outline in the text protocol. The MG1655 mutation rates of three different phenotypic markers, cycloserine, rifampicin, and nalidixic acid are shown here.
Mutation rates are assessed in Davis minimal medium with glucose concentration of 80 mg/L, 125 mg/L, and 250 mg/L. In one case, a glucose concentration of 1000 mg/L is used. As expected, mutation rates are higher for cycloserine resistance, lowest for nalidixic acid resistance, and the rate of rifampicin resistance is in the middle.
The fluctuation assay clearly shows which strain is a constitutive mutator and which has normal mutation rates. As the mutT deletant strain had a mutation rate to nalidixic acid resistance that is approximately 50x higher than the control MG1655 strain. While preforming this protocol, make sure that your cells are growing well.
They should be growing to uniformed density across parallel cultures with the same environment. One follow up to this procedure is to determine the DNA sequence of the rpoB gene in resistant colonies. To determine how the spectrum of mutations to rifampicin resistance varies with environmental microbial genotype.
Fluctuation assays in the 20th century demonstrated how mutation is spontaneous and random with respects to the environment. Now, they're shedding new light on how mutation rates do vary with the environment genetically, ecologically, even socially.
