The expression of a gene to a protein is one of the most vital biological processes. The use of light to control biology offers unparalleled spatiotemporal resolution from an external, orthogonal signal. A variety of methods have been developed that use light to control the steps of transcription and translation of specific genes into proteins, for cell-free to in vivo biotechnology applications. These methods employ techniques ranging from the modification of small molecules, nucleic acids and proteins with photocages, to the engineering of proteins involved in gene expression using naturally light-sensitive proteins. Although the majority of currently available technologies employ ultraviolet light, there has been a recent increase in the use of functionalities that work at longer wavelengths of light, to minimise cellular damage and increase tissue penetration. Here, we discuss the different chemical and biological methods employed to control gene expression, while also highlighting the central themes and the most exciting applications within this diverse field.

We report a single‐molecule mechanistic investigation into 2‐cyanobenzothiazole (CBT) chemistry within a protein nanoreactor. When simple thiols reacted reversibly with CBT, the thioimidate monoadduct was approximately 80‐fold longer‐lived than the tetrahedral bisadduct, with important implications for the design of molecular walkers. Irreversible condensation between CBT derivatives and N‐terminal cysteine residues has been established as a biocompatible reaction for site‐selective biomolecular labeling and imaging. During the reaction between CBT and aminothiols, we resolved two transient intermediates, the thioimidate and the cyclic precursor of the thiazoline product, and determined the rate constants associated with the stepwise condensation, thereby providing critical information for a variety of applications, including the covalent inhibition of protein targets and dynamic combinatorial chemistry.

Gene-expressing compartments assembled from simple, modular parts, are a versatile platform for creating minimal synthetic cells. In a manner synonymous with natural cells, these life-like assemblies utilise information encoded in DNA within the compartment's interior to dictate which proteins are expressed and consequently the overall function of the synthetic cell. Hence, by incorporating gene regulatory motifs into the DNA templates, in situ gene expression can be controlled according to specific stimuli. In this work, cell-free protein synthesis within synthetic cells was controlled using light by encoding genes of interest on light-activated DNA templates. Light-activated DNA contained a photocleavable blockade within the T7 promoter region that tightly repressed transcription until the blocking groups were removed with UV light. In this way, synthetic cells were activated remotely, in a spatiotemporally controlled manner. By applying this strategy to the expression of an acyl homo-serine lactone synthetase, BjaI, quorum-sensing based communication between synthetic cells and bacteria was controlled with light. This work provides a framework for the remote-controlled production and delivery of small molecules from non-living matter to living matter, with applications in biology and medicine.

Gene-expressing compartments assembled from simple, modular parts, are a versatile platform for creating minimal synthetic cells with life-like functions. By incorporating gene regulatory motifs into their encapsulated DNA templates, in situ gene expression and, thereby, synthetic cell function can be controlled according to specific stimuli. In this work, cell-free protein synthesis within synthetic cells was controlled using light by encoding genes of interest on light-activated DNA templates. Light-activated DNA contained a photocleavable blockade within the T7 promoter region that tightly repressed transcription until the blocking groups were removed with ultraviolet light. In this way, synthetic cells were activated remotely, in a spatiotemporally controlled manner. By applying this strategy to the expression of an acyl homoserine lactone synthase, BjaI, quorum-sensing-based communication between synthetic cells and bacteria was controlled with light. This work provides a framework for the remote-controlled production and delivery of small molecules from nonliving matter to living matter, with applications in biology and medicine.

Cell-free gene expression is a vital research tool to study biological systems in defined minimal environments and has promising applications in biotechnology. Developing methods to control DNA templates for cell-free expression will be important for precise regulation of complex biological pathways and use with synthetic cells, particularly using remote, non-damaging stimuli such as visible light. Here, we have synthesised blue light-activatable DNA parts that tightly regulate cell-free RNA and protein synthesis. We found that this blue light-activated DNA could initiate expression orthogonally to our previously generated UV light-activated DNA, which we used to generate a dual-wavelength light-controlled cell-free AND-gate. By encapsulating these orthogonal light-activated DNAs into synthetic cells, we used two overlapping patterns of blue and UV light to provide precise spatiotemporal control over the logic gate. Our blue and UV orthogonal light-activated DNAs will open the door for precise control of cell-free systems in biology and medicine.