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).