A synthetic multicellular system for programmed pattern formation.

Pattern formation is a hallmark of coordinated cell behaviour in both single and multicellular organisms. It typically involves cell-cell communication and intracellular signal processing. Here we show a synthetic multicellular system in which genetically engineered 'receiver' cells are programmed to form ring-like patterns of differentiation based on chemical gradients of an acyl-homoserine lactone (AHL) signal that is synthesized by 'sender' cells. In receiver cells, 'band-detect' gene networks respond to user-defined ranges of AHL concentrations. By fusing different fluorescent proteins as outputs of network variants, an initially undifferentiated 'lawn' of receivers is engineered to form a bullseye pattern around a sender colony. Other patterns, such as ellipses and clovers, are achieved by placing senders in different configurations. Experimental and theoretical analyses reveal which kinetic parameters most significantly affect ring development over time. Construction and study of such synthetic multicellular systems can improve our quantitative understanding of naturally occurring developmental processes and may foster applications in tissue engineering, biomaterial fabrication and biosensing.

Synthetic biology: new engineering rules for an emerging discipline.

Synthetic biologists engineer complex artificial biological systems to investigate natural biological phenomena and for a variety of applications. We outline the basic features of synthetic biology as a new engineering discipline, covering examples from the latest literature and reflecting on the features that make it unique among all other existing engineering fields. We discuss methods for designing and constructing engineered cells with novel functions in a framework of an abstract hierarchy of biological devices, modules, cells, and multicellular systems. The classical engineering strategies of standardization, decoupling, and abstraction will have to be extended to take into account the inherent characteristics of biological devices and modules. To achieve predictability and reliability, strategies for engineering biology must include the notion of cellular context in the functional definition of devices and modules, use rational redesign and directed evolution for system optimization, and focus on accomplishing tasks using cell populations rather than individual cells. The discussion brings to light issues at the heart of designing complex living systems and provides a trajectory for future development.

Engineering enzyme specificity using computational design of a defined-sequence library.

Engineered biosynthetic pathways have the potential to produce high-value molecules from inexpensive feedstocks, but a key limitation is engineering enzymes with high activity and specificity for new reactions. Here, we developed a method for combining structure-based computational protein design with library-based enzyme screening, in which inter-residue correlations favored by the design are encoded into a defined-sequence library. We validated this approach by engineering a glucose 6-oxidase enzyme for use in a proposed pathway to convert D-glucose into D-glucaric acid. The most active variant, identified after only one round of diversification and screening of only 10,000 wells, is approximately 400-fold more active on glucose than is the wild-type enzyme. We anticipate that this strategy will be broadly applicable to the discovery of new enzymes for engineered biological pathways.

Spatiotemporal control of gene expression with pulse-generating networks.

One of the important challenges in the emerging field of synthetic biology is designing artificial networks that achieve coordinated behavior in cell communities. Here we present a synthetic multicellular bacterial system where receiver cells exhibit transient gene expression in response to a long-lasting signal from neighboring sender cells. The engineered sender cells synthesize an inducer, an acyl-homoserine lactone (AHL), which freely diffuses to spatially proximate receiver cells. The receiver cells contain a pulse-generator circuit that incorporates a feed-forward regulatory motif. The circuit responds to a long-lasting increase in the level of AHL by transiently activating, and then repressing, the expression of a GFP. Based on simulation models, we engineered variants of the pulse-generator circuit that exhibit different quantitative responses such as increased duration and intensity of the pulse. As shown by our models and experiments, the maximum amplitude and timing of the pulse depend not only on the final inducer concentration, but also on its rate of increase. The ability to differentiate between various rates of increase in inducer concentrations affords the system a unique spatiotemporal behavior for cells grown on solid media. Specifically, receiver cells can respond to communication from nearby sender cells while completely ignoring communication from senders cells further away, despite the fact that AHL concentrations eventually reach high levels everywhere. Because of the resemblance to naturally occurring feed-forward motifs, the pulse generator can serve as a model to improve our understanding of such systems.