Synthetic gene networks have wide-ranging uses in reprogramming and rewiring organisms. To date, there has not been a way to harness the vast potential of these networks beyond the constraints of a laboratory or in vivo environment. Here, we present an in vitro paper-based platform that provides a new venue for synthetic biologists to operate, and a much-needed medium for the safe deployment of engineered gene circuits beyond the lab. Commercially available cell-free systems are freeze-dried onto paper, enabling the inexpensive, sterile and abiotic distribution of synthetic biology-based technologies for the clinic, global health, industry, research and education. For field use, we create circuits with colorimetric outputs for detection by eye, and fabricate a low-cost, electronic optical interface. We demonstrate this technology with small molecule and RNA actuation of genetic switches, rapid prototyping of complex gene circuits, and programmable in vitro diagnostics, including glucose sensors and strain-specific Ebola virus sensors.

The ability to rationally engineer microorganisms has been a long-envisioned goal dating back more than a half-century. With the genomics revolution and rise of systems biology in the 1990s came the development of a rigorous engineering discipline to create, control and programme cellular behaviour. The resulting field, known as synthetic biology, has undergone dramatic growth throughout the past decade and is poised to transform biotechnology and medicine. This Timeline article charts the technological and cultural lifetime of synthetic biology, with an emphasis on key breakthroughs and future challenges.

Biocontainment systems that couple environmental sensing with circuit-based control of cell viability could be used to prevent escape of genetically modified microbes into the environment. Here we present two engineered safe-guard systems: the Deadman and Passcode kill switches. The Deadman kill switch uses unbalanced reciprocal transcriptional repression to couple a specific input signal with cell survival. The Passcode kill switch uses a similar two-layered transcription design and incorporates hybrid LacI/GalR family transcription factors to provide diverse and complex environmental inputs to control circuit function. These synthetic gene circuits efficiently kill Escherichia coli and can be readily reprogrammed to change their environmental inputs, regulatory architecture and killing mechanism.

The cell poles constitute key subcellular domains that are often critical for motility, chemotaxis, and chromosome segregation in rod-shaped bacteria. However, in nearly all rods, the processes that underlie the formation, recognition, and perpetuation of the polar domains are largely unknown. Here, in Vibrio cholerae, we identified HubP (hub of the pole), a polar transmembrane protein conserved in all vibrios, that anchors three ParA-like ATPases to the cell poles and, through them, controls polar localization of the chromosome origin, the chemotactic machinery, and the flagellum. In the absence of HubP, oriCI is not targeted to the cell poles, chemotaxis is impaired, and a small but increased fraction of cells produces multiple, rather than single, flagella. Distinct cytoplasmic domains within HubP are required for polar targeting of the three ATPases, while a periplasmic portion of HubP is required for its localization. HubP partially relocalizes from the poles to the mid-cell prior to cell division, thereby enabling perpetuation of the polar domain in future daughter cells. Thus, a single polar hub is instrumental for establishing polar identity and organization.

Tunable control of protein degradation in bacteria would provide a powerful research tool. We use components of the Mesoplasma florum tmRNA system to create a synthetic degradation system that provides both independent control of the steady-state protein level and inducible degradation of targeted proteins in Escherichia coli. We demonstrate application of this system in synthetic circuit development and control of core bacterial processes and antibacterial targets, and transfer the system to Lactococcus lactis to establish its broad functionality in bacteria. We create a 238-member library of tagged essential proteins in E. coli that can serve as both a research tool to study essential gene function and an applied system for antibiotic discovery. Our synthetic protein degradation system is modular, does not require disruption of host systems, and can be transferred to diverse bacteria with minimal modification.

Vibrio cholerae is a human pathogen that causes the life-threatening diarrheal disease cholera. A type VI secretion system (T6SS) was recently shown to be required for full virulence in the O37 serogroup strain V52, which causes only sporadic human disease, but T6SS is not expressed in seventh pandemic O1 El Tor strains under standard laboratory conditions. In this study, we show that in the O1 El Tor strain C6706, T6SS is repressed by both quorum sensing and the uncharacterized protein VC0070 (TsrA). Disruption of TsrA and the quorum sensing regulator LuxO induces expression and secretion of the T6SS substrate Hcp, and this is dependent on the downstream regulator HapR, which directly binds to the promoter region of the T6SS genes hcp1 and hcp2 to induce expression. The activated T6SS in C6706 is functional and can translocate the effector protein VgrG-1 into macrophage cells, and T6SS activation leads to fecal diarrhea and intestinal inflammation in infant rabbits. Using an infant mouse infection model, we show that deletion of tsrA results in a 9.3-fold increase in intestinal colonization compared with wild type. TsrA functions as a global regulator to activate expression of hemagglutinin protease and repress cholera toxin and toxin coregulated pilus. Our findings provide significant insight into the molecular mechanism of T6SS and ToxT regulon gene regulation by quorum sensing and TsrA.

No abstract

Microbiota-modulating interventions are an emerging strategy to promote gastrointestinal homeostasis. Yet, their use in the detection, prevention, and treatment of acute infections remains underexplored. We report the basis of a probiotic-based strategy to promote colonization resistance and point-of-need diagnosis of cholera, an acute diarrheal disease caused by the pathogen Vibrio cholerae. Oral administration of Lactococcus lactis, a common dietary fermentative bacterium, reduced intestinal V. cholerae burden and improved survival in infected infant mice through the production of lactic acid. Furthermore, we engineered an L. lactis strain that specifically detects quorum-sensing signals of V. cholerae in the gut and triggers expression of an enzymatic reporter that is readily detected in fecal samples. We postulate that preventive dietary interventions with fermented foods containing natural and engineered L. lactis strains may hinder cholera progression and improve disease surveillance in populations at risk of cholera outbreaks.