Research in our group focuses on understanding the rules of evolution. With this project, we've developed a new protocol to study how thermophilic microbes evolve using controlled laboratory experiments. This will let us answer questions like how they respond to environmental change through adaptive evolution.
A major challenge is controlling cultivation conditions. Thermophiles require high-temperature environments for growth, leading to high evaporation rates and the risk of dried cultures and growth plates over the incubation period. Another challenge is the slow growth rates of some thermophiles, which can make rapid iteration testing challenging.
Our protocol addresses the major challenges associated with cultivating thermophilic microbes at multiple temperatures. We now have a better ability to control environmental conditions, ensuring the results we observe are consistent and reflect the experimental conditions we impose. This will enable us to study thermophile adaptation in real time.
Our protocol offers a high-throughput method, not just for Sulfolobus research, but adaptable to a variety of microorganisms. Using thermomixers, we can carry out simultaneous studies at different temperatures without requiring multiple large shaking incubators. It also reduces energy costs, offering a greener pathway for this type of research.
Our findings pave the way for evolution experiments in Sulfolobus and other thermophiles. Much of what we think we know about evolution comes from studying mesophilic organisms, and there's a risk that we're missing the key rules from thermophiles that will help us explain how the diversity of life on Earth has evolved. To begin, use a Bunsen burner to carefully heat a blunt steel wire.
Pierce it into the lid of a 2 ml microcentrifuge tube to create a 1 mm wide hole. Place the pierced tubes in an autoclavable vessel for sterilization. Once the tubes have been autoclaved and cooled, fill them with 1.5 ml of pre-warmed BBM+Now use a pipette tip to scrape about 50 to 60 mg of the Sulfolobus acidocaldarius culture from a glycerol stock.
Transfer the inoculum into the BBM+filled tubes. Keep an uninoculated tube with BBM+as the negative control. To prevent any aerosols from escaping from the pierced tube lid, place a heat-tolerant gas-permeable membrane on the top of the lid.
Place the inoculated tubes in a thermomixer for inoculation. After the cultures have reached the required optical density, centrifuge the tubes at 5, 000 g for two minutes at room temperature, then pipette out the supernatant to discard it. Add 1.5 ml of warm BBM+to the pellet and resuspend the cell pellet in the medium.
The growth was found to be similar when comparing incubation using thermomixers with that in conventional incubators. To begin, use a spectrophotometer to measure the optical density of a Sulfolobus acidocaldarius starting culture. Next, create serial dilutions of the starting culture from 10 to 1 to 10 to 6.
Pipette 100 microliters each from the 10 to 5 and 10 to 6 dilutions, then transfer them into the wells of a six-well plate containing solid BBM+Place the plate in a box with damp cloth to a static incubator at 75 degrees Celsius for five to seven days until single colonies emerge. To establish the desired number of evolving lineages from single colonies, with an inoculation loop, pick a colony at random. Resuspend the colony in 1.5 ml of pre-warmed BBM+contained in a pierced 2 ml tube.
Label the tube appropriately. After repeating the resuspension for several colonies, fill a tube with 1.5 ml of the medium as negative control. Seal the tops of the tubes with a breathable membrane, then incubate them in a thermomixer until the cultures become turbid.
Centrifuge the clonal cultures at 5, 000 g for one minute at room temperature. After discarding the supernatant, pipette 200 microliters of BBM+into the tube and resuspend the pellet. Add 200 microliters of 50%glycerol to each tube to create glycerol stocks.
Then store the stocks at 80 degrees Celsius until further experimentation. To begin, use the revived ancestral Sulfolobus acidocaldarius populations. Add BBM+to dilute the cultures to an OD600 of 0.01.
Pipette appropriate volume from each of the seven cultures into three separate pierced 2 ml tubes. Seal the tubes with a breathable membrane. Then place each set of seven ancestral populations into separate thermomixers.
Set each thermomixer to the required temperature and 400 RPM to begin the evolution experiment. After 48 hours, transfer 15 microliters of each of the transfer-zero cultures into 1.5 ml of warmed BBM+medium contained in a pierced 2 ml tube labeled Transfer 1. Seal the tubes with a permeable membrane before placing them back in randomized positions into the respective thermomixers.
Measure the OD600 of transfer-zero in a plate-based spectrophotometer to complete the first transfer. Repeat the culture transfer and measurement of optical density every two days to perform transfers until the desired number of transfers is reached. Prepare glycerol stocks of populations after every 10th transfer and on the final transfer, and label the tube with the ancestral population identifier and the transfer number.
Thermomixers should be set to constant temperatures of 75 degrees Celsius and 65 degrees Celsius. A third thermomixer will be gradually decreased to observe the response to gradual change. To begin, incubate the revived ancestral and temperature-evolved cultures of Sulfolobus acidocaldarius at the final temperatures.
Once incubation is complete, measure their optical densities at 600 nanometers. Next, add 1.5 ml of BBM+into six sets of seven tubes for each culture. Then add 15 microliters of each culture into the six sets of tubes.
Set three thermomixers to 75 degrees Celsius and three thermomixers to 65 degrees Celsius. Place the tubes in the thermomixer corresponding to the temperature and replicate ID.After a 48-hour incubation, transfer 200 microliters from each culture into a 96-well plate. Measure the optical density with a spectrophotometer.
Finally, plot and analyze the data with statistical software. Lineages from the constant 75-degree Celsius condition increased in OD600 from an initial range by the end of the experiment. In contrast, lineages from the constant 65-degree Celsius treatment displayed a drop.
Populations in the temperature-drop treatment increased from the initial optical density to Transfer 6, then showed a steady decline.
