Cell-free microcompartmentalised transcription-translation for the prototyping of synthetic communication networks

Abstract

Recent efforts in synthetic biology have shown the possibility of engineering distributed functions in populations of living cells, which requires the development of highly orthogonal, genetically encoded communication pathways. Cell-free transcription-translation (TXTL) reactions encapsulated in microcompartments enable prototyping of molecular communication channels and their integration into engineered genetic circuits by mimicking critical cell features, such as gene expression, cell size, and cell individuality within a community. In this review, we discuss the uses of cell-free transcription-translation reactions for the development of synthetic genetic circuits, with a special focus on the use of microcompartments supporting this reaction. We highlight several studies where molecular communication between non-living microcompartments and living cells have been successfully engineered.

Graphical abstract

Figure 1

Cell-free reactions for characterising, testing, and optimising complex circuits. Cell-free TXTL reactions can be used in combination with mathematical modelling to test complex circuits, and identify optimal conditions for the implementation of novel biological functions in vivo(a). Cell-free reactions are used to characterise the behaviour of novel isolated circuit parts [19^••] (upper panel (b)) and test the behaviour of circuits combining novel parts (lower panel (b)). Various versions of similar networks can rapidly be tested in TXTL, which can be used to reveal retroactive interactions between modules (upper panel (c)), as well as unexpected effects when combining components into a single construct (lower panel (c)). Cell burden occurs when a synthetic circuit excessively mobilises resources that are also necessary for endogenous circuits (left panel (d)). A capacity monitor reports possible cell burden during the implementation of novel circuits using TXTL and allows the design of networks generating minimal burden [21] (right panel (d)). TXTL allows the testing of various biochemical conditions, such as cofactor, salt, and enzyme concentrations, in order to optimise a TXTL-based reaction [30] (e).

Figure 2

Microfluidic technologies for studying gene expression at the cellular scale. Continuous flow reactors allow the implementation of out-of-equilibrium gene networks in TXTL. Valves precisely control addition and mixing of fresh TXTL reagents, enabling the implementation of complex networks such as oscillators [19^••] (a). Control over geometry and diffusion allows the study of gene expression propagation and pattern formation in compartmentalised flow reactors using DNA brushes [41] (b). Microdroplets generated on a microchip allow the screening of a vast range of conditions influencing network behaviour in TXTL, such as DNA template concentrations, shown here for the implementation of an incoherent feedforward loop [44] (c). Liposomes encapsulating TXTL can be obtained by double-emulsion techniques ((d), top-left panel). Liposomes are used for isolating genetic cascades [75] ((d), bottom-left panel) and creating units containing self-replicating genetic information [53^••] ((d), right panel). Upon increase of ionic strength, TXTL mixture can phase separate, resulting in a highly active microcompartment [47] ((e) left panel). DNA-functionalised hydrogels are used to create membraneless compartments capable of gene expression ((e) right panel).

Figure 3

TXTL in liposomes for prototyping novel communication channels. Through the exchange of chemical signalling molecules, liposomes containing TXTL mixture and quorum sensing circuits can be used as artificial cells to establish communication with other artificial cells (a) or with living cells (b).