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Showing papers by "Ron Weiss published in 2005"


Journal ArticleDOI
28 Apr 2005-Nature
TL;DR: 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 signal that is synthesized by ‘sender” cells is shown.
Abstract: 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.

1,165 citations


Journal ArticleDOI
TL;DR: The construction of synthetic transcriptional cascades comprising one, two, and three repression stages are reported, which enable us to analyze sensitivity and noise propagation as a function of network complexity.
Abstract: The precise nature of information flow through a biological network, which is governed by factors such as response sensitivities and noise propagation, greatly affects the operation of biological systems. Quantitative analysis of these properties is often difficult in naturally occurring systems but can be greatly facilitated by studying simple synthetic networks. Here, we report the construction of synthetic transcriptional cascades comprising one, two, and three repression stages. These model systems enable us to analyze sensitivity and noise propagation as a function of network complexity. We demonstrate experimentally steady-state switching behavior that becomes sharper with longer cascades. The regulatory mechanisms that confer this ultrasensitive response both attenuate and amplify phenotypical variations depending on the system's input conditions. Although noise attenuation allows the cascade to act as a low-pass filter by rejecting short-lived perturbations in input conditions, noise amplification results in loss of synchrony among a cell population. The experimental results demonstrating the above network properties correlate well with simulations of a simple mathematical model of the system.

543 citations


Journal ArticleDOI
TL;DR: Two artificial cell-cell communication systems in yeast were developed and analyzed and integrated Arabidopsis thaliana signal synthesis and receptor components with yeast endogenous protein phosphorylation elements and new response promoters resulted in population density–dependent gene expression and quorum sensing.
Abstract: The construction of synthetic cell-cell communication networks can improve our quantitative understanding of naturally occurring signaling pathways and enhance our capabilities to engineer coordinated cellular behavior in cell populations. Towards accomplishing these goals in eukaryotes, we developed and analyzed two artificial cell-cell communication systems in yeast. We integrated Arabidopsis thaliana signal synthesis and receptor components with yeast endogenous protein phosphorylation elements and new response promoters. In the first system, engineered yeast 'sender' cells synthesize the plant hormone cytokinin, which diffuses into the environment and activates a hybrid exogenous/endogenous phosphorylation signaling pathway in nearby engineered yeast 'receiver' cells. For the second system, the sender network was integrated into the receivers under positive-feedback regulation, resulting in population density-dependent gene expression (that is, quorum sensing). The combined experimental work and mathematical modeling of the systems presented here can benefit various biotechnology applications for yeast and higher level eukaryotes, including fermentation processes, biomaterial fabrication and tissue engineering.

200 citations


Journal ArticleDOI
TL;DR: As the techniques for system design, synthesis and optimization mature, synthetic systems will witness a rapid growth in the capabilities of synthetic systems with a wide-range of applications.

163 citations


Journal ArticleDOI
TL;DR: A technique for detecting weak transcriptional responses using signal-amplifying genetic circuits is presented and previously undetectable log phase responses of several Rhl quorum sensing controlled (qsc) promoters from Pseudomonas aeruginosa are revealed.
Abstract: Small changes in transcriptional activity often significantly affect phenotype but are not detectable in vivo by conventional means. To address this problem, we present a technique for detecting weak transcriptional responses using signal-amplifying genetic circuits. We apply this technique to reveal previously undetectable log phase responses of several Rhl quorum sensing controlled (qsc) promoters from Pseudomonas aeruginosa. Genetic circuits with Rhl promoters and transcriptional amplification components were built and tested in Escherichia coli. This enabled us to isolate the behavior of the promoters under study from Las and quinolone interactions. To amplify qsc promoter responses to acyl-homoserine lactones (AHL), the highly efficient lambda repressor gene was placed downstream of several Rhl promoters and coupled to a fluorescent reporter under the control of the lambda P(R) promoter. With amplification, up to approximately 100-fold differences in fluorescence levels between AHL induced and noninduced cultures were observed for promoters whose responses were otherwise not detectable. In addition, the combination of using signal amplification and performing experiments in E. coli simplified the analysis of AHL signal crosstalk. For example, we discovered that while a C4HSL/RhlR complex activates both qscrhlA and qscphzA1, a 3OC12HSL/RhlR complex activates qscphzA1 but not qscrhlA in our system. This crosstalk information is particularly important since one of the potential uses of amplification constructs is for the detection of specific quorum sensing signals in environmental and clinical isolates. Furthermore, the process of decomposing networks into basic parts, isolating these components in a well-defined background, and using amplification to characterize both crosstalk and cognate signal responses embodies an important approach to understanding complex genetic networks.

59 citations


Proceedings ArticleDOI
18 Mar 2005
TL;DR: The results using simulated data indicate that the algorithm was able to provide better parameter estimates for the pulse generating network than for the transcriptional cascade, and the variation in the magnitudes of the standard deviations between parameter estimates may give an indication of system sensitivity to specific kinetic rate constants.
Abstract: In this paper, we use two synthetic gene networks, a transcriptional cascade and a pulse generating network, to study the efficacy of a simple statistical parameter fitting algorithm. The fitting was performed on experimental data and computer-generated data (to test how well the algorithm works under ideal conditions with perfect information). Most of the experimental parameter estimations yielded tight ranges of kinetic values for both gene networks. However, the results using simulated data indicate that the algorithm was able to provide better parameter estimates for the pulse generating network than for the transcriptional cascade. This is likely a result of the larger amount of time-series data available for the pulse generator and its greater level of phenotypical complexity, leading to tighter constraints for optimization. The variation in the magnitudes of the standard deviations between parameter estimates may give an indication of system sensitivity to specific kinetic rate constants. In the future, we also plan to verify the experimental results by constructing network variants and attempting to predict behaviors using values obtained in this study.

22 citations


Proceedings ArticleDOI
08 Jun 2005
TL;DR: The design and simulation of a synthetic multi-cellular maze-solving system programmed to use artificial cell-to-cell communication and regulatory feedback in order to illuminate the correct path in a user-defined maze of cells arranged on a surface is presented.
Abstract: Control system theory provides convenient tools and concepts for describing and analyzing complex cell functions. In this paper we demonstrate the use of control theory to forward-engineer a complex synthetic gene network constructed from several modular components. Specifically, we present the design and simulation of a synthetic multi-cellular maze-solving system. Here, bacterial cells are programmed to use artificial cell-to-cell communication and regulatory feedback in order to illuminate the correct path in a user-defined maze of cells arranged on a surface. Simulations were used to analyze the system's spatiotemporal dynamics and sensitivity to various kinetic parameters. Experiments with Escherichia coli were carried out to characterize the diffusion properties of artificial cell-to-cell communication based on bacterial quorum sensing systems. The rational design process and simulation tools employed in this study provide an example for future engineering of complex synthetic gene networks comprising multiple control system motifs.

4 citations


Proceedings ArticleDOI
08 Aug 2005
TL;DR: Characterization of this simple network in mammalian cells is an important first step as this circuit will serve as a basis for building more complex networks that can select between many outputs using only a few inputs to form structures that resemble complex tissues like the spinal cord.
Abstract: Precise control of stem cell differentiation offers tremendous potential for tissue engineering. Synthetic gene networks provide a framework for understanding and engineering life. We propose to use synthetic gene networks to engineer circuits that dictate the cell fate of embryonic stem (ES) cells by controlling gene expression. These networks will be capable of turning on cell fate regulator genes in stem cells at precise times and under well-controlled and well-defined conditions based on external stimuli and the internal state of the cell. The over-expression of these cell fate regulator genes is sufficient to trigger particular differentiation path ways in ES cells. We have implemented a lentivirus delivered 1:2 multiplexer circuit for programmed differentiation. This network uses a transactivator, a repressor and one small molecule input Doxycycline (Dox). Dox selects to activate one of two cell fate regulator gene outputs, thereby pushing the ES cell along one of two differentiation pathways. Preliminary results demonstrate the ability to switch between the expression of two fluorescent proteins - EGFP and DsRed-Express based on the external input Dox. Upon integration of the cell fate regulators MyoD and Nanog into this circuit, ES cells will either differentiate into muscle or maintain their undifferentiated state. Characterization of this simple network in mammalian cells is an important first step as this circuit will serve as a basis for building more complex networks that can select between many outputs using only a few inputs to form structures that resemble complex tissues like the spinal cord.

1 citations


Proceedings ArticleDOI
08 Aug 2005
TL;DR: Results from directed evolution of receptor proteins to optimize receiver sensitivity and crosstalk interactions are presented, and simulations for two example pattern formation systems that can be constructed from these tuned components are presented.
Abstract: Biological pattern formation networks orchestrate complex processes of constituent cells, often through the use of multiple intercellular signals. The forward engineering of such multi-signal systems in synthetic biology has a number of important applications including biosensing, tissue engineering, and biomaterial fabrication. In addition, such synthetic systems provide a testing ground for quantitatively studying the fundamental principles governing similar natural genetic networks. However, an initial requirement for engineering multi-signal networks is the characterization and tuning of various properties of the signaling systems, including crosstalk, receiver response strength, and sensitivity. We characterize crosstalk interactions for synthetic receivers built from components of the Las and Rhl quorum sensing systems from Pseudomonas aeruginosa. Next, we present results from genetic constructs designed to amplify weak transcriptional responses to signaling molecules. We then discuss results from directed evolution of receptor proteins to optimize receiver sensitivity. These methods of engineering synthetic constructs with desired response strengths and sensitivities to external signals have a number of important applications in their own right, such as the development of biosensors for detection of trace amounts of toxins. In addition to the experimental results that show how signaling constructs can be optimized for such applications, we present simulations for two example pattern formation systems that can be constructed from these tuned components.

1 citations