Summary Biochemical systems in living cells have their optimum concentration ratio among each constituent element to maintain their functionality. However, in the case of the biochemical system with complex interactions and feedbacks among elements, their activity as a system greatly changes by the concentration shift of the entire system irrespective of the concentration ratio among elements. In this study, by using a transcription-translation (TX-TL) system as the subject, we illustrate the principle of the nonlinear relationship between the system concentration and the activity of the system. Our experiment and simulation showed that shifts of the system concentration of TX-TL by dilution and concentration works as a switch of activity and demonstrated its ability to induce a biochemical system to confer the permeability of small molecules to liposomes. These results contribute to the creation of artificial cells with the switch and provide an insight into the emergence of protocells.

Summary We present a protocol for activating protein synthesis in liposomes encapsulating a diluted E. coli cell extract-based TX-TL (transcription-translation) system by hypertonic concentration. Protein expression is turned on in the liposome-encapsulated TX-TL system by simple treatment with a concentrated external solution. The expression of sfGFP is demonstrated here, but it can be applied to other proteins. This protocol can be applied to the development of artificial cells utilizing the switch-on mechanism to activate protein expression, responding to the outer environment. For complete details on the use and execution of this protocol, please refer to Akui et al. (2021) .

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