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Publications about 'synthetic biology'
Articles in journal or book chapters
  1. V. H. Nagaraj, J. M. Greene, A. M. Sengupta, and and E.D. Sontag. Translation inhibition and resource balance in the TX-TL cell-free gene expression system. Synthetic Biology, 2017. Note: In press. Preprint in biorxiv 10.1101/142869 with same title, May 2017.Keyword(s): cell-free systems, in vitro synthetic biology.
    Abstract:
    Utilizing the synthetic transcription-translation (TX-TL) system, this paper studies the impact of nucleotide triphosphates (NTPs) and magnesium (Mg2+), on gene expression, in the context of the counterintuitive phenomenon of suppression of gene expression at high NTP concentration. Measuring translation rates for different Mg2+ and NTP concentrations, we observe a complex resource dependence. We demonstrate that translation is the rate-limiting process that is directly inhibited by high NTP concentrations. Additional Mg2+ can partially reverse this inhibition. In several experiments, we observe two maxima of the translation rate viewed as a function of both Mg2+ and NTP concentration, which can be explained in terms of an NTP-independent effect on the ribosome complex and an NTP- Mg2+ titration effect. The non-trivial compensatory effects of abundance of different vital resources signals the presence of complex regulatory mechanisms to achieve optimal gene expression.


  2. T.H. Segall-Shapiro, E. D. Sontag, and C. A. Voigt. Constant gene expression at any copy number using feedforward stabilized promoters. Submitted to Nature Biotechnology, 2017. Keyword(s): synthetic biology, systems biology, genetic circuits, gene copy number.
    Abstract:
    This paper deals with the design of promoters that maintain constant levels of expression, whether they are carried at single copy in the genome or on high-copy plasmids. The design is based on an incoherent feedforward loop (iFFL) with a perfectly non-cooperative repression. The circuits are implemented in E. coli using Transcription Activator Like Effectors (TALEs). The resulting stabilized promoters generate near identical expression across different genome locations and plasmid backbones (pSC101, p15a, ColE1, pUC), and also provide robustness to strain mutations and growth media. Further, their strength is tunable and can be used to maintain constant ratios between proteins.


  3. T. Kang, R. Moore, Y. Li, E.D. Sontag, and L. Bleris. Discriminating direct and indirect connectivities in biological networks. Proc Natl Acad Sci USA, 112:12893-12898, 2015. [PDF] Keyword(s): modular response analysis, stochastic systems, reverse engineering, gene networks, synthetic biology.
    Abstract:
    Reverse engineering of biological pathways involves an iterative process between experiments, data processing, and theoretical analysis. In this work, we engineer synthetic circuits, subject them to perturbations, and then infer network connections using a combination of nonparametric single-cell data resampling and modular response analysis. Intriguingly, we discover that recovered weights of specific network edges undergo divergent shifts under differential perturbations, and that the particular behavior is markedly different between different topologies. Investigating topological changes under differential perturbations may address the longstanding problem of discriminating direct and indirect connectivities in biological networks.


  4. T.H. Segall-Shapiro, A.J. Meyer, A.D. Ellington, E.D. Sontag, and C.A. Voigt. A `resource allocator' for transcription based on a highly fragmented T7 RNA polymerase. Molecular Systems Biology, 10:742-, 2014. [WWW] [PDF] Keyword(s): systems biology, synthetic biology, gene expression.
    Abstract:
    A transcriptional system is built based on a 'resource allocator' that sets a core RNAP concentration, which is then shared by multiple sigma fragments, which provide specificity. Adjusting the concentration of the core sets the maximum transcriptional capacity available to a synthetic system.


  5. T. Kang, J.T. White, Z. Xie, Y. Benenson, E.D. Sontag, and L. Bleris. Reverse engineering validation using a benchmark synthetic gene circuit in human cells. ACS Synthetic Biology, 2:255-262, 2013. [PDF] Keyword(s): reverse engineering, systems biology, synthetic biology.
    Abstract:
    This work introduces an experimental platform customized for the development and verification of reverse engineering and pathway characterization algorithms in mammalian cells. Specifically, we stably integrate a synthetic gene network in human kidney cells and use it as a benchmark for validating reverse engineering methodologies. The network, which is orthogonal to endogenous cellular signaling, contains a small set of regulatory interactions that can be used to quantify the reconstruction performance. By performing successive perturbations to each modular component of the network and comparing protein and RNA measurements, we study the conditions under which we can reliably reconstruct the causal relationships of the integrated synthetic network.


  6. V. Shimoga, J.T. White, Y. Li, E.D. Sontag, and L. Bleris. Synthetic mammalian transgene negative autoregulation. Molecular Systems Biology, 9:670-, 2013. [PDF] Keyword(s): systems biology, synthetic biology, gene expression.
    Abstract:
    Using synthetic circuits stably integrated in human kidney cells, we study the effect of negative feedback regulation on cell-wide (extrinsic) and gene-specific (intrinsic) sources of uncertainty. We develop a theoretical approach to extract the two noise components from experiments and show that negative feedback reduces extrinsic noise while marginally increasing intrinsic noise, resulting to significant total noise reduction. We compare the results to simple negative regulation, where a constitutively transcribed transcription factor represses a reporter protein. We observe that the control architecture also reduces the extrinsic noise but results in substantially higher intrinsic fluctuations. We conclude that negative feedback is the most efficient way to mitigate the effects of extrinsic fluctuations by a sole regulatory wiring.


  7. M. Miller, M. Hafner, E.D. Sontag, N. Davidsohn, S. Subramanian, P. E. M. Purnick, D. Lauffenburger, and R. Weiss. Modular design of artificial tissue homeostasis: robust control through synthetic cellular heterogeneity. PLoS Computational Biology, 8:e1002579-, 2012. [PDF] Keyword(s): systems biology, homeostasis, stem cells, synthetic biology.
    Abstract:
    Synthetic biology efforts have largely focused on small engineered gene networks, yet understanding how to integrate multiple synthetic modules and interface them with endogenous pathways remains a challenge. Here we present the design, system integration, and analysis of several large scale synthetic gene circuits for artificial tissue homeostasis. Diabetes therapy represents a possible application for engineered homeostasis, where genetically programmed stem cells maintain a steady population of beta-cells despite continuous turnover. We develop a new iterative process that incorporates modular design principles with hierarchical performance optimization targeted for environments with uncertainty and incomplete information. We employ theoretical analysis and computational simulations of multicellular reaction/diffusion models to design and understand system behavior, and find that certain features often associated with robustness (e.g., multicellular synchronization and noise attenuation) are actually detrimental for tissue homeostasis. We overcome these problems by engineering a new class of genetic modules for 'synthetic cellular heterogeneity' that function to generate beneficial population diversity. We design two such modules (an asynchronous genetic oscillator and a signaling throttle mechanism), demonstrate their capacity for enhancing robust control, and provide guidance for experimental implementation with various computational techniques. We found that designing modules for synthetic heterogeneity can be complex, and in general requires a framework for non-linear and multifactorial analysis. Consequently, we adapt a 'phenotypic sensitivity analysis' method to determine how functional module behaviors combine to achieve optimal system performance. We ultimately combine this analysis with Bayesian network inference to extract critical, causal relationships between a module's biochemical rate-constants, its high level functional behavior in isolation, and its impact on overall system performance once integrated.


  8. L. Bleris, Z. Xie, D. Glass, A. Adadey, E.D. Sontag, and Y. Benenson. Synthetic incoherent feed-forward circuits show adaptation to the amount of their genetic template. Molecular Systems Biology, 7:519-, 2011. [PDF] Keyword(s): adaptation, feedforward loops, systems biology, synthetic biology.
    Abstract:
    Natural and synthetic biological networks must function reliably in the face of fluctuating stoichiometry of their molecular components. These fluctuations are caused in part by changes in relative expression efficiency and the DNA template amount of the network-coding genes. Gene product levels could potentially be decoupled from these changes via built-in adaptation mechanisms, thereby boosting network reliability. Here we show that a mechanism based on an incoherent feed-forward motif enables adaptive gene expression in mammalian cells. We modeled, synthesized, and tested transcriptional and post-transcriptional incoherent loops and found that in all cases the gene product adapts to changes in DNA template abundance. We also observed that the post-transcriptional form results in superior adaptation behavior, higher absolute expression levels, and lower intrinsic fluctuations. Our results support a previously-hypothesized endogenous role in gene dosage compensation for such motifs and suggest that their incorporation in synthetic networks will improve their robustness and reliability.


  9. A.C. Jiang, A. C. Ventura, E. D. Sontag, S. D. Merajver, A. J. Ninfa, and D. Del Vecchio. Load-induced modulation of signal transduction networks. Science Signaling, 4, issue 194:ra67, 2011. [PDF] Keyword(s): systems biology, biochemical networks, synthetic biology, futile cycles, singular perturbations, modularity.
    Abstract:
    Biological signal transduction networks are commonly viewed as circuits that pass along in the process amplifying signals, enhancing sensitivity, or performing other signal-processing to transcriptional and other components. Here, we report on a "reverse-causality" phenomenon, which we call load-induced modulation. Through a combination of analytical and experimental tools, we discovered that signaling was modulated, in a surprising way, by downstream targets that receive the signal and, in doing so, apply what in physics is called a load. Specifically, we found that non-intuitive changes in response dynamics occurred for a covalent modification cycle when load was present. Loading altered the response time of a system, depending on whether the activity of one of the enzymes was maximal and the other was operating at its minimal rate or whether both enzymes were operating at submaximal rates. These two conditions, which we call "limit regime" and "intermediate regime," were associated with increased or decreased response times, respectively. The bandwidth, the range of frequency in which the system can process information, decreased in the presence of load, suggesting that downstream targets participate in establishing a balance between noise-filtering capabilities and a s ability to process high-frequency stimulation. Nodes in a signaling network are not independent relay devices, but rather are modulated by their downstream targets


  10. D. Del Vecchio and E.D. Sontag. Synthetic Biology: A Systems Engineering Perspective. In B.P. Ingalls and P. Iglesias, editors, Control Theory in Systems Biology, pages 101-123. MIT Press, 2009.
    Abstract:
    This is an expository paper about certain aspects of Synthetic Biology, including a discussion of the issue of modularity (load effects from downstream components).


  11. D. Del Vecchio and E.D. Sontag. Engineering Principles in Bio-Molecular Systems: From Retroactivity to Modularity. European Journal of Control, 15:389-397, 2009. Note: Preliminary version appeared as paper MoB2.2 in Proceedings of the European Control Conference 2009, August 23-26, 2009, Budapest. [PDF] Keyword(s): systems biology, biochemical networks, synthetic biology, futile cycles, singular perturbations, modularity.


  12. D. Del Vecchio, A.J. Ninfa, and E.D. Sontag. Modular Cell Biology: Retroactivity and Insulation. Molecular Systems Biology, 4:161, 2008. [PDF] Keyword(s): retroactivity, systems biology, biochemical networks, synthetic biology, futile cycles, singular perturbations, modularity.
    Abstract:
    Modularity plays a fundamental role in the prediction of the behavior of a system from the behavior of its components, guaranteeing that the properties of individual components do not change upon interconnection. Just as electrical, hydraulic, and other physical systems often do not display modularity, nor do many biochemical systems, and specifically, genetic networks. Here, we study the effect of interconnections on the input/output dynamic characteristics of transcriptional components, focusing on a property, which we call "retroactivity," that plays a role analogous to non-zero output impedance in electrical systems. In transcriptional networks, retroactivity is large when the amount of transcription factor is comparable to, or smaller than, the amount of promoter binding sites, or when the affinity of such binding sites is high. In order to attenuate the effect of retroactivity, we propose a feedback mechanism inspired by the design of amplifiers in electronics. We introduce, in particular, a mechanism based on a phosphorylation/dephosphorylation cycle. This mechanism enjoys a remarkable insulation property, due to the fast time scales of the phosphorylation and dephosphorylation reactions. Such a mechanism, when viewed as a signal transduction system, has thus an inherent capacity to provide insulation and hence to increase the modularity of the system in which it is placed.


Conference articles
  1. D. Del Vecchio, A.J. Ninfa, and E.D. Sontag. A Systems Theory with Retroactivity: Application to Transcriptional Modules. In Proceedings of the 2008 American Control Conference, Seattle, June 2008, pages Paper WeC04.1, 2008. [PDF] Keyword(s): retroactivity, systems biology, biochemical networks, synthetic biology, futile cycles, singular perturbations, modularity.


  2. D. Del Vecchio and E.D. Sontag. Dynamics and control of synthetic bio-molecular networks. In Proceedings American Control Conf., New York, July 2007, pages 1577-1588, 2007. Keyword(s): systems biology, biochemical networks, synthetic biology.
    Abstract:
    This tutorial paper presents an introduction to systems and synthetic molecular biology. It provides an introduction to basic biological concepts, and describes some of the techniques as well as challenges in the analysis and design of biomolecular networks.



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Last modified: Thu Nov 23 10:40:57 2017
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