Thermal Adaptive Laboratory Evolution (TALE) is a method used to help microbial cells adapt to high-temperature environments through gradual, lab-based evolution. Instead of altering genes directly, scientists grow cells under increasingly warm temperatures over many generations. Each round of growth selects for cells that are better at surviving and reproducing in heat. Over time, these evolutionary pressures lead to a genetically adapted, heat-tolerant strain. This technique is valuable in synthetic biology, where many engineered enzymes work best at higher temperatures than their host cells normally tolerate.

The process involved serial passaging of the minimal cells in liquid SP4 media, with a gradual increase in incubation temperature across multiple generations. Cells were initially cultured at 37°C, their native optimal temperature, and once growth was observed, small aliquots were transferred to fresh media and incubated at slightly higher temperatures (e.g., 38°C, 39°C, 40°C, etc.). This stepwise thermal selection continued over time, ultimately reaching 43.7°C. All culturing was performed inside a thermocycler, which allowed precise control of temperature conditions throughout the adaptation process. At each stage, only the cultures that showed healthy growth (pH indicator in culture turning yellow) were carried forward, effectively selecting for spontaneous mutations that supported thermal tolerance.