The overall goal of this procedure is to enable a microorganism to evolve in laboratory conditions. This method can help answer key questions in the microbiology field, such as adrenal function relationships, stress responses, metabolic engineering, and of course, evolution. The main advantage of this technique is continuous selection of the most suaded descendant under specific laboratory conditions.
Begin this procedure with the preparation of equipment, as well as initial medium, stress medium and high stress medium, as described in the text protocol. Inoculate a single colony or wild type E.coli in a 15-milliliter test tube containing four milliliters of initial medium. Incubate the test tube in a shaking incubator for 12 hours at 37 degrees celsius and 220 rpm.
Incubate the chemostat jar providing for aeration and agitation at 37 degrees celsius for six hours. Following the incubation, aseptically connect the end of the silicon tubing from the pumps to the chemostat jar. Aseptically transfer one milliliter of pre-culture to the chemostat jar.
Start the outlet pump and collect the culture in the exponential phase. Check the optical density at 600 nanometers of the culture from the outlet tubing. Then, start the inlet pump.
Check the optical density of the culture at 600 nanometers from the outlet tubing every 24 hours. After operating the chemostat for 96 hours, which is a 9.6 full turnover, exchange the reservoir to the lowest concentration of high stress medium. When you exchange the reservoir to a higher stress medium, the cells may be shocked.
If the cell density is too low, stop feeding the higher stress medium for some time to restore the cell density. If the optical density is lower than 0.2, stop the feeding inlet pump for six hours. Restart the inlet pump and check that the optical density is over 0.2.
Gradually increase the concentration of the stressor by incrementally changing to a reservoir containing a higher stressor concentration. Take samples of the adapted culture whenever it reaches a stressor adaptation milestone and store for further genomic analysis. For sample storage, mix the 0.5 milliliter culture sample with 0.5 milliliters of a sterilized 80%glycerol solution and store it at 80 degrees celsius.
Prepare 1.6%ager plate medium containing the same stressor at the same concentration as in the medium. Plate 0.1 milliliters of the outlet culture from the chemostat and incubate at 37 degrees celsius for 16 hours. Following incubation, pick single colonies from the plate using a sterile toothpick and inoculate them in 15 milliliter test tubes containing the same stressor and at the same medium concentration as in the chemostat.
Incubate for six hours. Transfer one milliliter of the culture broth into a 250 milliliter erlenmeyer flask containing 50 milliliters of medium. Harvest 0.5 milliliters of the culture broth every hour and measure the optical density at 600 nanometers.
Compare the growth rate of the adapted strain to that of the wild type strain given the stressor. The wild type E.coli strain was evolved in a high succinate stressed condition for 270 days, which corresponds to nearly 930 generations. Whenever higher succinate stress was added, the biomass concentration was immediately lowered and then restored.
Shown here is a schematic diagram of the chemostat with a mutant that is tolerant against the high succinate stress. Most wild type cells are washed out under the stress conditions and the mutant grows more. Eventually, the population of the mutant descendant is increased.
The second and third mutations are continuously selected for and eventually dominate the chemostat. This figure shows that the adapted strain grew without delay under high succinate stressed conditions, while the wild type strain did not. A similar strategy can be applied to adaptive laboratory evolution using a chemostat with a variation of stressors.
After watching this video, you should have a good understanding of how to acquire and evolve the microorganism under a specified condition. Once mastered, this technique can be done in a couple of month. While attempting this procedure, it's important to remember not to wash out chemostat cells by adding too much stress at once and remember to check the optical density every day.
Following this procedure are the methods like a genome analysis and transcriptome analysis can be performed in order to answer additional questions like what mutation contributes to the stress tolerant evolution?
