Ron Weiss

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.

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.

Amorphous computing

ion to Continuous Space and Time The amorphous model postulates computing particles distributed throughout a space. If the particles are dense, one can imagine the particles as actually filling the space, and create programming abstractions that view the space itself as the object being programmed, rather than the collection of particles. Beal and Bachrach [10, 4] pursued this approach by creating a language, Proto, where programmers specify the behavior of an amorphous computer as though it were a continuous material filling the space it occupies. Proto programs manipulate fields of values spanning the entire space. Programming primitives are designed to make it simple to compile global operations to operations at each point of the continuum. These operations are approximated by having each device represent a nearby chunk of space. Programs are specified in space and time units that are independent of the distribution of particles and of the particulars of communication and execution on those particles (Figure 5). Programs are composed functionally, and many of the details of communication and composition are made implicit by Proto’s runtime system, allowing complex programs to be expressed simply. Proto has been applied to applications in sensor networks like target tracking and threat avoidance, to swarm robotics and to modular robotics, e.g., generating a planar wave for coordinated actuation. Newton’s language Regiment [45, 44] also takes a continuous view of space and time. Regiment is organized in terms of stream operations, where each stream represents a time-varying quantity over a part of space, for example, the average value of the temperature over a disc of a given radius centered at a designated point. Regiment, also a functional language, is designed to gather streams of data from regions of the amorphous computer and accumulate them at a single point. This assumption allows Regiment to provide region-wide summary functions that are difficult to implement in Proto.

Programmed population control by cell–cell communication and regulated killing

De novo engineering of gene circuits inside cells is extremely difficult, and efforts to realize predictable and robust performance must deal with noise in gene expression and variation in phenotypes between cells. Here we demonstrate that by coupling gene expression to cell survival and death using cell–cell communication, we can programme the dynamics of a population despite variability in the behaviour of individual cells. Specifically, we have built and characterized a ‘population control’ circuit that autonomously regulates the density of an Escherichia coli population. The cell density is broadcasted and detected by elements from a bacterial quorum-sensing system, which in turn regulate the death rate. As predicted by a simple mathematical model, the circuit can set a stable steady state in terms of cell density and gene expression that is easily tunable by varying the stability of the cell–cell communication signal. This circuit incorporates a mechanism for programmed death in response to changes in the environment, and allows us to probe the design principles of its more complex natural counterparts.

Multi-Input RNAi-Based Logic Circuit for Identification of Specific Cancer Cells

A synthetic biomolecular circuit identifies abnormal cell states by the integration of multiple endogenous microRNA inputs. Engineered biological systems that integrate multi-input sensing, sophisticated information processing, and precisely regulated actuation in living cells could be useful in a variety of applications. For example, anticancer therapies could be engineered to detect and respond to complex cellular conditions in individual cells with high specificity. Here, we show a scalable transcriptional/posttranscriptional synthetic regulatory circuit—a cell-type “classifier”—that senses expression levels of a customizable set of endogenous microRNAs and triggers a cellular response only if the expression levels match a predetermined profile of interest. We demonstrate that a HeLa cancer cell classifier selectively identifies HeLa cells and triggers apoptosis without affecting non-HeLa cell types. This approach also provides a general platform for programmed responses to other complex cell states.

Directed evolution of a genetic circuit

The construction of artificial networks of transcriptional control elements in living cells represents a new frontier for biological engineering. However, biological circuit engineers will have to confront their inability to predict the precise behavior of even the most simple synthetic networks, a serious shortcoming and challenge for the design and construction of more sophisticated genetic circuitry in the future. We propose a combined rational and evolutionary design strategy for constructing genetic regulatory circuits, an approach that allows the engineer to fine-tune the biochemical parameters of the networks experimentally in vivo. By applying directed evolution to genes comprising a simple genetic circuit, we demonstrate that a nonfunctional circuit containing improperly matched components can evolve rapidly into a functional one. In the process, we generated a library of genetic devices with a range of behaviors that can be used to construct more complex circuits.

HyPursuit: a hierarchical network search engine that exploits content-link hypertext clustering

HyPursuit is a new hierarchical network search engine that clusters hypertext documents to structure a given information space for browsing and search act ivities. Our content-link clustering algorithm is based on the semantic information embedded in hyperlink structures and document contents. HyPursuit admits multiple, coexisting cluster hierarchies based on different principles for grouping documents, such as the Library of Congress catalog scheme and automatically created hypertext clusters. HyPursuit’s abstraction functions summarize cluster contents to support scalable query processing. The abstraction functions satisfy system resource limitations with controlled information 10SS. The result of query processing operations on a cluster summary approximates the result of performing the operations on the entire information space. We constructed a prototype system comprising 100 leaf WorldWide Web sites and a hierarchy of 42 servers that route queries to the leaf sites. Experience with our system suggests that abstraction functions based on hypertext clustering can be used to construct meaningful and scalable cluster hierarchies. We are also encouraged by preliminary results on clustering based on both document contents and hyperlink structures.

Highly efficient Cas9-mediated transcriptional programming

The RNA-guided nuclease Cas9 can be reengineered as a programmable transcription factor. However, modest levels of gene activation have limited potential applications. We describe an improved transcriptional regulator obtained through the rational design of a tripartite activator, VP64-p65-Rta (VPR), fused to nuclease-null Cas9. We demonstrate its utility in activating endogenous coding and noncoding genes, targeting several genes simultaneously and stimulating neuronal differentiation of human induced pluripotent stem cells (iPSCs).

Dipole anisotropy in the COBE DMR first year sky maps

We present a determination of the cosmic microwave background dipole amplitude and direction from the COBE Differential Microwave Radiometers (DMR) first year of data. Data from the six DMR channels are consistent with a Doppler-shifted Planck function of dipole amplitude ΔT=3.365±0.027 mK toward direction (l II , b II )=(264°.4±0°.3, 48°.4±0°.5). The implied velocity of the Local Group with respect to the CMB rest frame is v LG =627±22 km s −1 toward (l II , b II )=(276°±3°, 30°±3°). DMR has also mapped the dipole anisotropy resulting from the Earths orbital motion about the Solar system barycenter, yielding a measurement of the monopole CMB temperature T 0 at 31.5, 53, and 90 GHz, T 0 =2.75±0.05 KWe present a determination of the cosmic microwave background dipole amplitude and direction from the COBE Differential Microwave Radiometers (DMR) first year of data. Data from the six DMR channels are consistent with a Doppler-shifted Planck function of dipole amplitude Delta T = 3.365 +/-0.027 mK toward direction (l,b) = (264.4 +/- 0.3 deg, 48.4 +/- 0.5 deg). The implied velocity of the Local Group with respect to the CMB rest frame is 627 +/- 22 km/s toward (l,b) = (276 +/- 3 deg, 30 +/- 3 deg). DMR has also mapped the dipole anisotropy resulting from the Earths orbital motion about the Solar system barycenter, yielding a measurement of the monopole CMB temperature at 31.5, 53, and 90 GHz, to be 2.75 +/- 0.05 K.

Engineered bidirectional communication mediates a consensus in a microbial biofilm consortium

Microbial consortia form when multiple species colocalize and communally generate a function that none is capable of alone. Consortia abound in nature, and their cooperative metabolic activities influence everything from biodiversity in the global food chain to human weight gain. Here, we present an engineered consortium in which the microbial members communicate with each other and exhibit a “consensus” gene expression response. Two colocalized populations of Escherichia coli converse bidirectionally by exchanging acyl-homoserine lactone signals. The consortium generates the gene-expression response if and only if both populations are present at sufficient cell densities. Because neither population can respond without the others signal, this consensus function can be considered a logical AND gate in which the inputs are cell populations. The microbial consensus consortium operates in diverse growth modes, including in a biofilm, where it sustains its response for several days.