Position-dependent gene expression in gradients of morphogens is one of the key processes involved in cellular differentiation during development. Here, we study a simple artificial differentiation process, which is based on the diffusion of genetic inducers within one-dimensional arrangements of 50 μm large water-in-oil droplets. The droplets are filled with either bacteria or cell-free gene expression systems, both equipped with genetic constructs that produce inducers or respond to them via expression of a fluorescent protein. We quantitatively study the coupled diffusion-gene expression process and demonstrate that gene expression can be made position-dependent both within bacteria-containing and cell-free droplets. By generating diffusing quorum sensing signals in situ, we also establish communication between artificial cell-free sender cells and bacterial receivers, and vice versa.

Lithographic patterning of DNA molecules enables spatial organization of cell-free genetic circuits under well-controlled experimental conditions. Here, we present a biocompatible, DNA-based resist termed "Bephore", which is based on commercially available components and can be patterned by both photo- and electron-beam lithography. The patterning mechanism is based on cleavage of a chemically modified DNA hairpin by ultraviolet light or electrons, and a subsequent strand-displacement reaction. All steps are performed in aqueous solution and do not require chemical development of the resist, which makes the lithographic process robust and biocompatible. Bephore is well suited for multistep lithographic processes, enabling the immobilization of different types of DNA molecules with micrometer precision. As an application, we demonstrate compartmentalized, on-chip gene expression from three sequentially immobilized DNA templates, leading to three spatially resolved protein-expression gradients.

CRISPR interference (CRISPRi) using dCas9-sgRNA is a powerful tool for the exploration and manipulation of gene functions. Here we quantify the reversible switching of a central process of the bacterial cell cycle by CRISPRi and an antisense RNA mechanism. Reversible induction of filamentous growth in E. coli has been recently demonstrated by controlling the expression levels of the bacterial cell division proteins FtsZ/FtsA via CRISPRi. If FtsZ falls below a critical level, cells cannot divide. However, the cells remain metabolically active and continue with DNA replication. We surmised that this makes them amenable to an inducible antisense RNA strategy to counteract FtsZ inhibition. We show that both static and inducible thresholds can adjust the characteristics of the switching process. Combining bulk data with single cell measurements, we characterize the efficiency of the switching process. Successful restoration of division is found to occur faster in the presence of antisense sgRNAs than upon simple termination of CRISPRi induction.

Molecular complexes composed of RNA molecules and proteins are promising multifunctional nanostructures for a wide variety of applications in biological cells or in artificial cellular systems. In this study, we systematically address some of the challenges associated with the expression and assembly of such hybrid structures using cell-free gene expression systems. As a model structure, we investigated a pRNA-derived RNA scaffold functionalized with four distinct aptamers, three of which bind to proteins, streptavidin and two fluorescent proteins, while one binds the small molecule dye malachite green (MG). Using MG fluorescence and Förster resonance energy transfer (FRET) between the RNA-scaffolded proteins, we assess critical assembly parameters such as chemical stability, binding efficiency, and also resource sharing effects within the reaction compartment. We then optimize simultaneous expression and coassembly of the RNA-protein nanostructure within a single-compartment cell-free gene expression system. We demonstrate expression and assembly of the multicomponent nanostructures inside of emulsion droplets and their aptamer-mediated localization onto streptavidin-coated substrates, plus the successful assembly of the hybrid structures inside of bacterial cells.