Fig. 2: Compartmentalization and feeding by a temperature difference.

a, A thermophoretic chamber filled with a TX-TL solution, expressing sfGFP at the steady state, was subjected to a temperature difference (27–40 °C). The accumulation of sfGFP was monitored by measuring fluorescence over time at different chamber positions (black squares), recording a 3-fold absolute and 25-fold relative accumulation (solid lines). The dashed lines correspond to a simulated finite element model (see the ‘Material and methods’ section in Supplementary Information). b, The setting was challenged by a continuous flow of water at the top of the chamber (11 µl h^–1; dark blue) to test the thermal confinement of sfGFP provided by the temperature difference. Without the temperature difference, diffusion into the outward flow slowly removed sfGFP from the chamber (grey arrows). The total pre-expressed sfGFP fluorescence remaining inside the chamber was modelled by finite element simulations for up to 6 months (dotted lines). The solid lines are averages of three experiments. As a comparison, \({{\rm{H}}}_{2}{{\rm{PO}}}_{4}^{-}\) was simulated as a possible waste molecule produced during sfGFP synthesis (large, dotted lines). \({{\rm{H}}}_{2}{{\rm{PO}}}_{4}^{-}\) was removed much faster from the thermal trap than sfGFP under isothermal conditions but also when a temperature gradient was present. c, Activation and retention of a deficient TX-TL reaction was probed by supplying the missing amino acids and tRNAs at a flow rate of 11 µl h^–1 at the top of the chamber. The sfGFP concentration was measured at the top and bottom of the chamber with and without applying a temperature difference. In isothermal conditions, the TX-TL reaction was first activated and then flushed out, leaving no synthesized sfGFP in the compartment in the long run. However, when a temperature difference was applied, the reaction was activated by the accumulated supply solution, leading to the synthesis and long-term accumulation and retention of sfGFP. d, Simulated kinetics of ATP (brown) and \({{\rm{H}}}_{2}{{\rm{PO}}}_{4}^{-}\) (blue) as feeding and waste molecules, respectively, in a simulated transcription reaction under an active thermal gradient. ATP was introduced in the trap at a concentration of 1 mM under the same fluid flow as in previous experiments and simulations. \({{\rm{H}}}_{2}{{\rm{PO}}}_{4}^{-}\), which is present in the trap before the start of the flow, is simultaneously removed from the trap by the continuous fluid flow at the top of the chamber and produced from ATP throughout the trap corresponding to the concentration of the TX-TL reaction. A stable state is reached after 800 h, in which \({{\rm{H}}}_{2}{{\rm{PO}}}_{4}^{-}\) is produced at the same rate as it is removed. On the other hand, ATP is continuously accumulating in the chamber and retained against the fluid flow.