Rapidly characterizing the fast dynamics of RNA genetic circuitry with cell-free transcription-translation (TX-TL) systems

Takahashi, Melissa K.; Chappell, James; Hayes, Clarmyra A.; Sun, Zachary Z.; Singhal, Vipul; Spring, Kevin; Al‐Khabouri, Shaima; Fall, Christopher P.; Noireaux, Vincent; Murray, Richard M.; Lucks, Julius B.

doi:10.1101/003335

Cited by 31 publications

(44 citation statements)

References 48 publications

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“…Finally, RNA circuits have the potential to propagate signals much faster than protein circuits, since signal propagation speed is determined by the fast degradation rates of RNAs. This was recently demonstrated by Takahashi et al for a transcriptional cascade composed of two layered sRNA transcription repressors (Figure 3) [35].…”

Section: Rna-based Genetic Circuitsmentioning

confidence: 87%

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A renaissance in RNA synthetic biology: new mechanisms, applications and tools for the future

Chappell

Watters

Takahashi

et al. 2015

Current Opinion in Chemical Biology

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132

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Since our ability to engineer biological systems is directly related to our ability to control gene expression, a central focus of synthetic biology has been to develop programmable genetic regulatory systems. Researchers are increasingly turning to RNA regulators for this task because of their versatility, and the emergence of new powerful RNA design principles. Here we review advances that are transforming the way we use RNAs to engineer biological systems. First, we examine new designable RNA mechanisms that are enabling large libraries of regulators with protein-like dynamic ranges. Next, we review emerging applications, from RNA genetic circuits to molecular diagnostics. Finally, we describe new experimental and computational tools that promise to accelerate our understanding of RNA folding, function and design.

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Section: Rna-based Genetic Circuitsmentioning

confidence: 87%

“…( Figure 3 Legend Continued) shows an RNA-only transcriptional cascade composed of two orthogonal transcriptional repressors (attenuators) [35]. Since the circuit signal is propagated by RNA species, dynamics are fast with response times in cell-free transcription and translation (TX-TL) reactions on the order of 5 min.…”

Section: Rna-based Genetic Circuitsmentioning

confidence: 99%

A renaissance in RNA synthetic biology: new mechanisms, applications and tools for the future

Chappell

Watters

Takahashi

et al. 2015

Current Opinion in Chemical Biology

Self Cite

150

132

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“…As a result, new components can be added or synthesized and can be maintained at precise concentrations, while the chemical environment is easily monitored and sampled (Carlson et al, 2012;You and Zhang, 2013). From a prototyping perspective, cell-free systems are particularly well suited to rapid design-buildtest cycles because they do not require the re-engineering of genetic pathways in organisms with each design (Bujara et al, 2010;Chappell et al, 2015;Karig et al, 2012;Shin and Noireaux, 2012;Sun et al, 2014;Takahashi et al, 2014). There is also a high degree of flexibility to model individual enzyme kinetics, measure metabolite fluxes in multistep pathways, determine catalyst stability, study the effects of redox potential on pathway performance, and experimentally isolate many other process properties that are confounded in living organisms.…”

Section: Introductionmentioning

confidence: 99%

Lysate of engineered Escherichia coli supports high-level conversion of glucose to 2,3-butanediol

Kay

Jewett

2015

Metabolic Engineering

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Cell-free metabolic engineering (CFME) is emerging as a powerful approach for the production of target molecules and pathway debugging. Unfortunately, high cofactor costs, limited cofactor and energy regeneration, and low volumetric productivities hamper the widespread use and practical implementation of CFME technology. To address these challenges, we have developed a cell-free system that harnesses ensembles of catalytic proteins prepared from crude lysates, or extracts, of cells to fuel highly active heterologous metabolic conversions. As a model pathway, we selected conversion of glucose to 2,3-butanediol (2,3-BD), a medium level commodity chemical with many industrial applications. Specifically, we engineered a single strain of Escherichia coli to express three pathway enzymes necessary to make meso-2,3-BD (m2,3-BD). We then demonstrated that lysates from this strain, with addition of glucose and catalytic amounts of cofactors NAD+ and ATP, can produce m2,3-BD. Endogenous glycolytic enzymes convert glucose to pyruvate, the starting intermediate for m2,3-BD synthesis. Strikingly, with no strain optimization, we observed a maximal synthesis rate of m2,3-BD of 11.3 ± 0.1 g/L/h with a theoretical yield of 71% (0.36 g m2,3-BD/g glucose) in batch reactions. Titers reached 82 ± 8 g/L m2,3-BD in a 30 h fed-batch reaction. Our results highlight the ability for high-level co-factor regeneration in cell-free lysates. Further, they suggest exciting opportunities to use lysate-based systems to rapidly prototype metabolic pathways and carry out molecular transformations when bioconversion yields (g product/L), productivities (g product/L/h), or cellular toxicity limit commercial feasibility of whole-cell fermentation.

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“…Earlier studies in the area of in vitro synthetic biology and cell-free systems have made important contributions to our understanding of fundamental molecular biology and biochemistry, and more recently in the study of molecular switch dynamics and complex gene circuits (Hong et al, 2014; Karzburn et al, 2014; Sun et al, 2014, Takahashi et al, 2014). These efforts, however, have focused on solution phase reactions using fresh from frozen cell-free systems and often in liposomes with the goal of assembling artificial cells (Kumura et al, 2009; Kobori et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

Paper-Based Synthetic Gene Networks

Pardee¹,

Green²,

Ferrante³

et al. 2014

Cell

644

771

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Synthetic gene networks have wide-ranging uses in reprogramming and rewiring organisms. To date, there has not been a way to harness the vast potential of these networks beyond the constraints of a laboratory or in vivo environment. Here, we present an in vitro paper-based platform that provides a new venue for synthetic biologists to operate, and a much-needed medium for the safe deployment of engineered gene circuits beyond the lab. Commercially available cell-free systems are freeze-dried onto paper, enabling the inexpensive, sterile and abiotic distribution of synthetic biology-based technologies for the clinic, global health, industry, research and education. For field use, we create circuits with colorimetric outputs for detection by eye, and fabricate a low-cost, electronic optical interface. We demonstrate this technology with small molecule and RNA actuation of genetic switches, rapid prototyping of complex gene circuits, and programmable in vitro diagnostics, including glucose sensors and strain-specific Ebola virus sensors.

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Rapidly characterizing the fast dynamics of RNA genetic circuitry with cell-free transcription-translation (TX-TL) systems

Cited by 31 publications

References 48 publications

A renaissance in RNA synthetic biology: new mechanisms, applications and tools for the future

A renaissance in RNA synthetic biology: new mechanisms, applications and tools for the future

Lysate of engineered Escherichia coli supports high-level conversion of glucose to 2,3-butanediol

Paper-Based Synthetic Gene Networks

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