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Cell-free systems, as part of the synthetic biology field, have become a critical platform in biological studies. However, there is a lack of research into developing a switch for a dynamical control of the transcriptional and translational process. The optogenetic tool has been widely proven as an ideal control switch for protein synthesis due to its nontoxicity and excellent time-space conversion. Hence, in this study, a blue light-regulated two-component system named YF1/FixJ was incorporated into an Escherichia coli-based cell-free system to control protein synthesis. The corresponding cell-free system successfully achieved a 5-fold dynamic protein expression by blue light repression and 3-fold dynamic expression by blue light activation. With the aim of expanding the applications of cell-free synthetic biology, the cell-free blue light-sensing system was used to perform imaging, light-controlled antibody synthesis, and light-triggered artificial cell assembly. This study can provide a guide for further research into the field of cell-free optical sensing. Moreover, it will also promote the development of cell-free synthetic biology and optogenetics through applying the cell-free optical sensing system to synthetic biology education, biopharmaceutical research, and artificial cell construction.

An artificial cell is a simplified model of a living system, bringing breakthroughs into both basic life science and applied research. The bottom-up strategy instructs the construction of an artificial cell from nonliving materials, which could be complicated and interdisciplinary considering the inherent complexity of living cells. Although significant progress has been achieved in the past 2 decades, the area is still facing some problems, such as poor compatibility with complex bio-systems, instability, and low standardization of the construction method. In this review, we propose creating artificial cells through the integration of different functional modules. Furthermore, we divide the function requirements of an artificial cell into four essential parts (metabolism, energy supplement, proliferation, and communication) and discuss the present researches. Then we propose that the compartment and the reestablishment of the communication system would be essential for the reasonable integration of functional modules. Although enormous challenges remain, the modular construction would facilitate the simplification and standardization of an artificial cell toward a natural living system. This function-based strategy would also broaden the application of artificial cells and represent the steps of imitating and surpassing nature.

Cell-free protein synthesis has been developed as a critical platform in synthetic biology. Unlike the cell-based synthesis system, cell-free system activates transcriptional and translational mechanisms in vitro , and can control protein synthesis by artificially adding components or chemicals. However, the control method puts forward higher requirements in terms of accurate and non-toxic control, which cannot be achieved by chemical substances. For cell-free system, physical signal is a kind of ideal spatiotemporal control approach to replace chemical substances, realizing high accuracy with little side effect. Here we review the methods of using physical signals to control gene expression in cell-free systems, including studies based on light, temperature, electric field, and magnetic force. The transfer of these switches into cell-free system further expands the flexibility and controllability of the system, thus further expanding the application capability of cell-free systems. Finally, existing problems such as signal source and signal transmission are discussed, and future applications in pharmaceutical production, delivery and industrial production are further looked into.