Temperature Evolution Experimentation Using Sulfolobus acidocldarius

To begin, use the revived ancestral Sulfolobus acidocaldarius populations. Add BBM plus to dilute the cultures to an OD600 of 0.01. Pipette appropriate volume from each of the seven cultures into three separate pierced two milliliter 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 milliliters of warmed BBM plus medium contained in a pierced two milliliter tube labeled transfer one.

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.