Extended Data Fig. 3: A selection method of the evolved IDPs exhibiting different thermoresponsive phase behaviors.
From: Directed evolution of functional intrinsically disordered proteins
a. Schematics of the selection strategies for the evolution of synIDPs with hysteric behaviors. To identify synIDP variants exhibiting reversible thermoresponsive phase transitions, a three-round replica plating strategy was employed. Following the LCST- and UCST- selection described above, colonies from the first round that displayed temperature-dependent are considered potential candidates for reversible phase behavior. Each of the two induced plates incubated at 30 °C and 45 °C are then independently replicated onto fresh plates containing the same arabinose concentration but incubated under the opposite temperature condition for at least 12 h. This second round of selection aimes to assess the reversibility of the observed temperature-dependent growth phenotype, distinguishing reversible or irreversible phase transitions. A third round of replica plating is transferring colonies from the second set of plates back to the original temperature condition (30 °C or 45 °C). b. Evaluation of CFUs of DH5α under different temperature. Statistical significance was determined using two sample T-test. Data is represented as mean ± SD. n = 3 biological replicates. c. Representative confocal images of an rIDP, and an irIDP illustrating differences in thermal hysteresis. RLPWT and ELPWT serve as controls exhibiting reversible UCST- and LCST-type phase behavior, respectively. Scale bars represent 3 μm.
