Engineering signaling circuits using a cell-free synthetic biology approach

Unravelling the complex organization of molecular networks inside living cells is a key topic of both systems biology and synthetic biology. Simplified model systems have been engineered and constructed under controlled cell-free conditions with the goal of mimicking biological responses of intracellular circuits. These model systems reveal key principles of molecular programs that underlie the biological function of interest. Here, we first present an overview of key studies on cell-free biochemical modules that are able to emulate higherorder dynamics. Additionally, we discuss the effect of retroactivity, a phenomenon resulting from the interconnection of an upstream module to a downstream module. Importantly, while cell-free studies on molecular networks are often performed at high reactant concentrations in a well-stirred dilute environment, the cell’s interior is an inhomogeneous crowded environment where reactions between biomolecules occur at low concentrations. We additionally discuss the stochastic nature of cellular reactions resulting from low concentrations of reactants and the effect of macromolecular crowding on biochemical reactions. Finally, we present recent work showing the versatility of programmable biochemical reaction networks in analytical and diagnostic applications. The work in this chapter has partly been published in: Hendrik W.H. van Roekelǂ, Bas J.H.M. Rosierǂ, Lenny H.H. Meijerǂ, Peter A.J. Hilbers, Albert J. Markvoort, Wilhelm T.S. Huck and Tom F.A. de Greef – Chemical Society Reviews, 2015, 44, 7465 ǂ contributed equally to this work