Highly modular bow-tie gene circuits with programmable dynamic behaviour

Abstract

Synthetic gene circuits often require extensive mutual optimization of their components for successful operation, while modular and programmable design platforms are rare. A possible solution lies in the “bow-tie” architecture, which stipulates a focal component - a “knot” - uncoupling circuits’ inputs and outputs, simplifying component swapping, and introducing additional layer of control. Here we construct, in cultured human cells, synthetic bow-tie circuits that transduce microRNA inputs into protein outputs with independently programmable logical and dynamic behavior. The latter is adjusted via two different knot configurations: a transcriptional activator causing the outputs to track input changes reversibly, and a recombinase-based cascade, converting transient inputs into permanent actuation. We characterize the circuits in HEK293 cells, confirming their modularity and scalability, and validate them using endogenous microRNA inputs in additional cell lines. This platform can be used for biotechnological and biomedical applications in vitro, in vivo, and potentially in human therapy.

Engineering Gene Circuits for Mammalian Cell–Based Applications

“…When integrated into gene circuits in a bowtie topology, recombinases can act as a "knot" to transmit the circuit's information and thereby improve the performance of gene circuits significantly by reducing component leakage and input and output decoupling, as well as the timely production of individual components induced by gene flipping (Fig. 3C) (Prochazka et al 2014). The production of an output protein within gene circuits is used to wire different gene switches within the circuit or to have a convenient readout protein for output quantification.…”

“…Circuit topology can also be applied in biology to show synthetic biological networks and to describe input -output functions. Gene circuits with different topologies, such as oscillators (Tigges et al 2009), logic gates (Kramer et al 2004;Rinaudo et al 2007;Smolke 2007, 2008;Ausländer et al 2012aAusländer et al , 2014a, hysteresis (Kramer and Fussenegger 2005), band-pass (Greber and Fussenegger 2010), time delay Lapique and Benenson 2014), bow-tie (Prochazka et al 2014), and memory devices (Burrill et al 2012) have already been constructed in living mammalian cells. Because the above-described synthetic gene switches are able to process input information and to produce a specific output, they are well-suited building blocks for the design of programmable gene circuits reminiscent of electronic circuits.…”

“…In particular, a GPCR-based CO 2 / H þ -inducible gene-regulation system was combined with bacterial repressor-and protein dimerization -based systems to build gas-inducible gene circuits. Gene circuits have also been designed to detect siRNA states in mammalian cells (Rinaudo et al 2007;Tigges et al 2009;Leisner et al 2010;Xie et al 2011;Lapique and Benenson 2014;Prochazka et al 2014). siRNA or miRNA target sites are integrated into synthetic components to process siRNA information logically, enabling either the programming of the resulting gene circuit by the exogenous application of siRNA molecules, the intracellular, ligand-inducible production of siRNA ligands or endogenous miRNA species.…”

“…Site-specific recombinases, which have the ability to excise, flip, or invert DNA sequences, have been established as powerful building blocks for genome engineering and transcriptional gene control in mammalian cells (Nern et al 2011). Site-specific recombination sites are engineered to flank genes, promoters, or transcriptional blockers to influence gene expression and to introduce regulatory knots to decouple inputs and outputs (Lapique and Benenson 2014;Prochazka et al 2014). When integrated into gene circuits in a bowtie topology, recombinases can act as a "knot" to transmit the circuit's information and thereby improve the performance of gene circuits significantly by reducing component leakage and input and output decoupling, as well as the timely production of individual components induced by gene flipping (Fig.…”

“…The design of such complex gene circuits is challenging because they must function precisely and specifically in the complicated cellular environment without influencing essential endogenous processes. For example, gene circuits are designed to improve, tune, or adapt the switching performance of target genes (Deans et al 2007;Lapique and Benenson 2014;Prochazka et al 2014) or to perform specific tasks such as biocomputing (Xie et al 2011;Ausländer et al 2012a) and oscillatory functions (Tigges et al 2009), as well as recording environmental and endogenous events in synthetic memory devices (Burrill et al 2012). Moreover, engineered cellcell communication devices enable mammalian cells to interact and assemble synthetic multicellular systems (Bacchus et al 2012).…”