An in silico modeling toolbox for rapid prototyping of circuits in a biomolecular &amp;#x201C;breadboard&amp;#x201D; system

Tuza, Zoltán A.; Singhal, Vipul; Kim, Jongmin; Murray, Richard M.

doi:10.1109/cdc.2013.6760079

Cited by 25 publications

(34 citation statements)

References 28 publications

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“…Throughout our experiments, we observed these effects of resource depletion, beginning at t0 ≈ 2 hours onwards. To be consistent with the modeling approaches of Tuza and Singhal, (Siegal-Gaskins et al, 2014;Tuza et al, 2013), we suppose that the translation rate kT L(t) decays with time as a first order process, beginning at time t0, and with decay parameter α d = log(2)/(480)…”

Section: Rfp Cfp Reporter Modelsmentioning

confidence: 98%

The Effect of Compositional Context on Synthetic Gene Networks

Yeung

Martin

et al. 2016

Preprint

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SUMMARYIt is well known that synthetic gene expression is highly sensitive to how comprising genetic elements (promoter structure, spacing regions between promoter and coding sequences, ribosome binding sites, etc.) are spatially configured. An important topic that has received far less attention is how the physical layout of entire genes within a synthetic gene network affects their individual expression levels. In this paper we show, both quantitatively and qualitatively, that compositional context can significantly alter expression levels in synthetic gene networks. We also show that these compositional context effects are pervasive both at the transcriptional and translational level. Further, we demonstrate that key characteristics of gene induction, such as ultrasensitivity and dynamic range, are heavily dependent on compositional context. We postulate that supercoiling can be used to explain these interference effects and validate this hypothesis through modeling and a series of in vitro supercoiling relaxation experiments. On the whole, these results suggest that compositional context introduces feedback in synthetic gene networks. As an illustrative example, we show that a design strategy incorporating compositional context effects can improve threshold detection and memory properties of the toggle switch.

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Section: Rfp Cfp Reporter Modelsmentioning

confidence: 98%

The Effect of Compositional Context on Synthetic Gene Networks

Yeung

Martin

et al. 2016

Preprint

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“…The blue and orange line are the respective predicted mRNA dynamics generated by the TX-TL modeling toolbox. 35 The linear DNA encoding other pathway enzymes competes with linear DNA encoding ald (C) for transcription. The mRNA expression difference of the peak mRNA value is greater than 80%.…”

Section: Transcriptionmentioning

confidence: 99%

System-level studies of a cell-free transcription-translation platform for metabolic engineering

Sato

Huang

et al. 2017

Preprint

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Current methods for assembling biosynthetic pathways in microorganisms require a process of repeated trial and error and have long design-build-test cycles. We describe the use of a cell-free transcription-translation (TX-TL) system as a biomolecular breadboard for the rapid engineering of the 1,4-butanediol (BDO) pathway. We demonstrate the reliability of TX-TL as a platform for engineering biological systems by undertaking a careful characterization of its transcription and translation capabilities and provide a detailed analysis of its metabolic output. Using TX-TL to survey the design space of the BDO pathway enables rapid tuning of pathway enzyme expression levels for improved product yield. Leveraging TX-TL to screen enzyme variants for improved catalytic activity accelerates design iterations that can be directly applied to in vivo strain development. IntroductionUtilizing fast-growing microorganisms to produce molecules of industrial relevance has the potential to rapidly advance the progress of green chemistry. Processes of traditional chemical synthesis require heavy metal catalysts, toxic solvents, and fossil fuels as feedstocks. The biosynthetic approach, which uses naturally occurring enzymes, less energy, and renewable feedstocks, is becoming an attractive alternative. 1-2 However, biosynthetic approaches are challenged by long design-build-test cycles. Microbial pathway engineering often has about oneweek cycle time. 3 The performance of the pathway is frequently far from design, requiring many iterations. 4 For example, it took DuPont and Genencor more than 100 person-years of work to develop the commercialization of biobased 1,3-propanediol. 5 Recent advances in cell-free systems offer an alternative to this costly approach. Cell-free systems have been used to reduce the cycle time of pathway construction. The design-build-test cycle in a cell-free system using linear DNA takes less than one day. 6

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“…Amino acids were assumed to be in excess so were not explicitly considered. As in previous models of cell-free reactions, consumption of ATP and GTP during translation was not considered (18). The translation reaction produced non-fluorescent immature GFP and this was assumed to mature in a first-order reaction into the detectable fluorescent species, GFP mat .…”

Section: Equationmentioning

confidence: 99%

“…We have combined this new ITT system with factorial experimental design, high-throughput automation via acoustic liquid handling robotics and Bayesian model parameter inference methods to create a platform to enable the rigorous and rapid characterization of novel genetic parts. A significant advantage of cell-free systems is that the defined reaction conditions permit the accurate characterization of genetic parts using sophisticated modelling techniques (7,8,18,19). However, such approaches have previously generally been applied to Escherichia coli gene expression circuits.…”

Section: Introductionmentioning

confidence: 99%

Prototyping ofBacillus megateriumgenetic elements through automated cell-free characterization and Bayesian modelling

MacDonald

Weinecke

Kylilis

et al. 2016

Preprint

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Automation and factorial experimental design together with cell-free in vitro transcription-translation systems offers a new route to the precise characterization of regulatory components. This now presents a new opportunity to illuminate the genetic circuitry from arcane microbial chassis, which are difficult to assess in vivo. One such host, Bacillus megaterium, is a giant microbe with industrial potential as a producer of recombinant proteins at gram per litre scale. Herein, we establish a B. megaterium cell-free platform and characterize a refactored xylose-repressor circuit using acoustic liquid handling robotics to simultaneously monitor 324 reactions in vitro. To accurately describe the system, we have applied a Bayesian statistical approach to infer model parameters by simultaneously using information from multiple experimental conditions. These developments now open up a new approach for the rapid and accurate characterization of genetic circuitry using cell-free reactions from unusual microbial cell chasses for bespoke applications.

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An in silico modeling toolbox for rapid prototyping of circuits in a biomolecular “breadboard” system

Cited by 25 publications

References 28 publications

The Effect of Compositional Context on Synthetic Gene Networks

The Effect of Compositional Context on Synthetic Gene Networks

System-level studies of a cell-free transcription-translation platform for metabolic engineering

Prototyping ofBacillus megateriumgenetic elements through automated cell-free characterization and Bayesian modelling

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