System concentration shift as a regulator of transcription-translation system within liposomes

Abstract

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.

Activation of a diluted E. coli cell-free transcription-translation system within liposomes by hypertonic concentration

“…Other genes should be expressed by the protocol. We have confirmed the expression of α-hemolysin by the protocol ( Akui et al., 2021 ). For the experimental condition, mix 20 μL of 2× External buffer with 20 μL of liposome solution in a 1.7 mL tube by gently pipetting.…”

“… Transform E. coli BL21 (DE3) with pET29-mCherry-His ( Akui et al., 2021 ) using competent cells, and cultivate overnight (16–20 h) at 37°C on LB agar medium supplemented with 50 μg/mL kanamycin at final. …”

“…Tens to hundreds of liposomes are collected by the protocol, and at least 72% liposomes show synthesis of sfGFP in cytosols (see also Akui et al, 2021 ). Typical data obtained by the protocol is shown in Figure 4 .…”

“…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) .…”

“…For complete details on the use and execution of this protocol, please refer to Akui et al. (2021) .…”