Synthetic biology has advanced the design of genetic devices that can be used to reprogram metabolic activities in mammalian cells. By functionally linking the signal transduction of melanopsin to the control circuit of the nuclear factor of activated T cells, we have designed a synthetic signaling cascade enabling light-inducible transgene expression in different cell lines grown in culture or bioreactors or implanted into mice. In animals harboring intraperitoneal hollow-fiber or subcutaneous implants containing light-inducible transgenic cells, the serum levels of the human glycoprotein secreted alkaline phosphatase could be remote-controlled with fiber optics or transdermally regulated through direct illumination. Light-controlled expression of the glucagon-like peptide 1 was able to attenuate glycemic excursions in type II diabetic mice. Synthetic light-pulse-transcription converters may have applications in therapeutics and protein expression technology.

Heterologous mammalian gene regulation systems for adjustable expression of multiple transgenes are necessary for advanced human gene therapy and tissue engineering, and for sophisticated in vivo gene-function analyses, drug discovery, and biopharmaceutical manufacturing. The antibiotic-dependent interaction between the repressor (E) and operator (ETR) derived from an Escherichia coli erythromycin-resistance regulon was used to design repressible (E(OFF)) and inducible (E(ON)) mammalian gene regulation systems (E.REX) responsive to clinically licensed macrolide antibiotics (erythromycin, clarithromycin, and roxithromycin). The E(OFF) system consists of a chimeric erythromycin-dependent transactivator (ET), constructed by fusing the prokaryotic repressor E to a eukaryotic transactivation domain that binds and activates transcription from ETR-containing synthetic eukaryotic promoters (P(ETR)). Addition of macrolide antibiotic results in repression of transgene expression. The E(ON) system is based on E binding to artificial ETR-derived operators cloned adjacent to constitutive promoters, resulting in repression of transgene expression. In the presence of macrolides, gene expression is induced. Control of transgene expression in primary cells, cell lines, and microencapsulated human cells transplanted into mice was demonstrated using the E.REX (E(OFF) and E(ON)) systems. The macrolide-responsive E.REX technology was functionally compatible with the streptogramin (PIP-regulated and tetracycline (TET-regulated expression systems, and therefore may be combined for multiregulated multigene therapeutic interventions in mammalian cells and tissues.

In multicellular systems cell identity is imprinted by epigenetic regulation circuits, which determine the global transcriptome of adult cells in a cell phenotype-specific manner. By combining two repressors, which control each other's expression, we have developed a mammalian epigenetic circuitry able to switch between two stable transgene expression states after transient administration of two alternate drugs. Engineered Chinese hamster ovary cells (CHO-K1) showed toggle switch-specific expression profiles of a human glycoprotein in culture, as well as after microencapsulation and implantation into mice. Switch dynamics and expression stability could be predicted with mathematical models. Epigenetic transgene control through toggle switches is an important tool for engineering artificial gene networks in mammalian cells.

Synthetic biology has shown that the metabolic behavior of mammalian cells can be altered by genetic devices such as epigenetic and hysteretic switches, timers and oscillators, biocomputers, hormone systems and heterologous metabolic shunts. To explore the potential of such devices for therapeutic strategies, we designed a synthetic mammalian circuit to maintain uric acid homeostasis in the bloodstream, disturbance of which is associated with tumor lysis syndrome and gout. This synthetic device consists of a modified Deinococcus radiodurans-derived protein that senses uric acids levels and triggers dose-dependent derepression of a secretion-engineered Aspergillus flavus urate oxidase that eliminates uric acid. In urate oxidase-deficient mice, which develop acute hyperuricemia, the synthetic circuit decreased blood urate concentration to stable sub-pathologic levels in a dose-dependent manner and reduced uric acid crystal deposits in the kidney. Synthetic gene-network devices providing self-sufficient control of pathologic metabolites represent molecular prostheses, which may foster advances in future gene- and cell-based therapies.

Intercellular communication within an organism, between populations, or across species and kingdoms forms the basis of many ecosystems in which organisms coexist through symbiotic, parasitic, or predator-prey relationships. Using multistep airborne communication and signal transduction, we present synthetic ecosystems within a mammalian cell population, in mice, or across species and kingdoms. Inter-and intrakingdom communication was enabled by using sender cells that produce volatile aldehydes, small vitamin-derived molecules, or antibiotics that diffuse, by gas or liquid phase, to receiver cells and induce the expression of specific target genes. Intercellular and cross-kingdom communication was shown to enable quorum sensing between and among mammalian cells, bacteria, yeast, and plants, resulting in precise spatiotemporal control of IFN-␤ production. Interconnection of bacterial, yeast, and mammalian cell signaling enabled the construction of multistep signal transduction and processing networks as well as the design of synthetic ecosystems that mimic fundamental coexistence patterns in nature, including symbiosis, parasitism, and oscillating predator-prey interactions.biological circuit ͉ gene switch ͉ synthetic biology I ntercellular cross-talk either within or between organisms of the same or different species represents a fundamental communication process that is responsible not only for orchestrating vital functions within multicellular life forms but also for determining the manner in which different organisms coexist. Whereas the most sophisticated intercellular communication networks manage processes such as cellular differentiation and pattern formation during development or use hormonal and neural circuitries to adapt responses by individual organisms to endogenous and exogenous stimuli, higher-order ecosystems are organized by basic interaction patterns known as symbiosis, parasitism, or predator-prey interactions. Pioneering advances in the design and study of synthetic multicellular systems have focused entirely on cross-talk within the same species. Quorum-sensing prokaryotic variants, engineered to produce lactones and broadcast this cell-density signal across a population, have been used to establish automated population control (1), programmed pattern formation (2, 3), or metabolic information processing in bacteria (4). Similar intrapopulation communication has also been successfully engineered in yeast using Arabidopsis thaliana-derived signaling pathways (5). Although these monospecies networks probe the design principles of their more complex natural counterparts, their extrapolation to mammalian cell communities or interspecies and interkingdom cross-talk remains limited. Furthermore, the use of nonvolatile small-molecule inducers as the broadcast signal in existing synthetic intercellular cross-talk systems necessitates that both sender and receiver cells reside in the same liquid environment. Sender cells transgenic for expression of alcohol dehydrogenase (ADH) produced acetaldehyde, ...