An engineered mammalian band-pass network

Abstract

Gene expression circuitries, which enable cells to detect precise levels within a morphogen concentration gradient, have a pivotal impact on biological processes such as embryonic pattern formation, paracrine and autocrine signalling, and cellular migration. We present the rational synthesis of a synthetic genetic circuit exhibiting band-pass detection characteristics. The components, involving multiply linked mammalian trans-activator and -repressor control systems, were selected and fine-tuned to enable the detection of ‘low-threshold’ morphogen (tetracycline) concentrations, in which target gene expression was triggered, and a ‘high-threshold’ concentration, in which expression was muted. In silico predictions and supporting experimental findings indicated that the key criterion for functional band-pass detection was the matching of componentry that enabled sufficient separation of the low and high threshold points. Using the circuitry together with a fluorescence-encoded target gene, mammalian cells were genetically engineered to be capable of forming a band-like pattern of differentiation in response to a tetracycline chemical gradient. Synthetic gene networks designed to emulate naturally occurring gene behaviours provide not only insight into biological processes, but may also foster progress in future tissue engineering, gene therapy and biosensing applications.

A three-step framework for programming pattern formation

“…FF systems are possibly the most studied of all patterning mechanisms and have featured heavily in synthetic biology engineering projects [5][6][7][8][9][10][11][12]. The so-called "band detectors" draw their inspiration from well-characterised biological systems ( Table 1).…”

“…They have been implemented artificially in vitro (e.g. 'Drosophila-on-a chip [5]), as well as in vivo in prokaryotic [6][7][8]10,12,13] and eukaryotic organisms [11]. Synthetic interactions have been programmed at the Protein-DNA [7][8][9][10]13], RNA-RNA [10] and Protein-Protein [10] levels.…”

Engineering Gene Circuits for Mammalian Cell–Based Applications

“…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.…”

Application of Synthetic Biology to Regenerative Medicine

“…Greber et al [44] constructed a synthetic genetic network with band-pass detection characteristics; output expression is 'off' where there is no signal, 'on' across a window of low concentrations of input but 'off' again when the input concentration is high. Such a system is very interesting from the point of view of tissue engineering because it can be used for spatial patterning.…”

“…We expect this list of mammalian bio-parts to grow rapidly in the coming years as other elegant prokaryotic designs (genetic counter [44], which is based on two separate pathways that can each inhibit the production of the output molecule. The top pathway, on its own a high-pass filter, makes molecule A to repress the production of output molecule unless tetracycline (tet) is present at high enough concentrations to inhibit production of A.…”