A synthetic gene circuit is often engineered by considering the host cell as an invariable "chassis". Circuit activation, however, may modulate host physiology, which in turn can drastically impact circuit behavior. We illustrate this point by a simple circuit consisting of mutant T7 RNA polymerase (T7 RNAP*) that activates its own expression in bacterium Escherichia coli. Although activation by the T7 RNAP* is noncooperative, the circuit caused bistable gene expression. This counterintuitive observation can be explained by growth retardation caused by circuit activation, which resulted in nonlinear dilution of T7 RNAP* in individual bacteria. Predictions made by models accounting for such effects were verified by further experimental measurements. Our results reveal a novel mechanism of generating bistability and underscore the need to account for host physiology modulation when engineering gene circuits.

Biological research is experiencing an increasing focus on the application of knowledge rather than on its generation. Thanks to the increased understanding of cellular systems and technological advances, biologists are more frequently asking not only 'how can I understand the structure and behaviour of this biological system?', but also 'how can I apply that knowledge to generate novel functions in different biological systems or in other contexts?' Active pursuit of the latter has nurtured the emergence of synthetic biology. Here, we discuss the motivation behind, and foundational technologies enabling, the development of this nascent field. We examine some early successes and applications while highlighting the challenges involved. Finally, we consider future directions and mention non-scientific considerations that can influence the field's growth.

Synthetic biology seeks to enable programmed control of cellular behavior though engineered biological systems. These systems typically consist of synthetic circuits that function inside, and interact with, complex host cells possessing pre-existing metabolic and regulatory networks. Nevertheless, while designing systems, a simple well-defined interface between the synthetic gene circuit and the host is frequently assumed. We describe the generation of robust but unexpected oscillations in the densities of bacterium Escherichia coli populations by simple synthetic suicide circuits containing quorum components and a lysis gene. Contrary to design expectations, oscillations required neither the quorum sensing genes (luxR and luxI) nor known regulatory elements in the PluxI promoter. Instead, oscillations were likely due to density-dependent plasmid amplification that established a population-level negative feedback. A mathematical model based on this mechanism captures the key characteristics of oscillations, and model predictions regarding perturbations to plasmid amplification were experimentally validated. Our results underscore the importance of plasmid copy number and potential impact of “hidden interactions” on the behavior of engineered gene circuits - a major challenge for standardizing biological parts. As synthetic biology grows as a discipline, increasing value may be derived from tools that enable the assessment of parts in their final context.

Protein thermodynamic stability is a fundamental physical characteristic that determines biological function. Furthermore, alteration of thermodynamic stability by macromolecular interactions or biochemical modifications is a powerful tool for assessing the relationship between protein structure, stability, and biological function. High-throughput approaches for quantifying protein stability are beginning to emerge that enable thermodynamic measurements on small amounts of material, in short periods of time, and using readily accessible instrumentation. Here we present such a method, fast quantitative cysteine reactivity (fQCR), which exploits the linkage between protein stability, sidechain protection by protein structure, and structural dynamics to characterize the thermodynamic and kinetic properties of proteins. In this approach, the reaction of a protected cysteine and thiol-reactive fluorogenic indicator is monitored over a gradient of temperatures after a short incubation time. These labeling data can be used to determine the midpoint of thermal unfolding, measure the temperature dependence of protein stability, quantify ligand-binding affinity, and, under certain conditions, estimate folding rate constants. Here, we demonstrate the fQCR method by characterizing these thermodynamic and kinetic properties for variants of Staphylococcal nuclease and E. coli ribose-binding protein engineered to contain single, protected cysteines. These straightforward, information-rich experiments are likely to find applications in protein engineering and functional genomics.