Cell-Free Translation Is More Variable than Transcription

Chizzolini, Fabio; Forlin, Michele; Martín, Noël Yeh; Berloffa, Giuliano; Cecchi, Dario; Mansy, Sheref S.

doi:10.1021/acssynbio.6b00250

Cited by 42 publications

(56 citation statements)

References 49 publications

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“…Having identified that translation initiation is inefficient in PUREfrex, we next attempted to improve this process. We first hypothesized that translation initiation is hampered by mRNA secondary structures (58,(77)(78)(79)(80) that would be more susceptible to form in vitro due to poor coupling between transcription and translation. In E. coli, the first ribosome translating an mRNA often closely follows the transcribing RNA polymerase (see Castro-Roa and Zenkin (81) for an in vitro study with the endogenous E. coli RNA polymerase).…”

Section: Attempts To Restore Transcription-translation Coupling Failementioning

confidence: 99%

Modelling cell-free RNA and protein synthesis with minimal systems

et al. 2019

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DNA-guided cell-free protein synthesis using a minimal set of purified components has emerged as a versatile platform in constructive biology. The E. coli-based PURE (Protein synthesis Using Recombinant Elements) system offers the basic protein synthesis factory in a prospective minimal cell relying on extant molecules. However, it becomes urgent to improve the system's performance, and to build a mechanistic computational model that can help interpret and predict gene expression dynamics. Herein, we utilized all three commercially available PURE system variants: PURExpress, PUREfrex and PUREfrex2.0. We monitored apparent kinetics of mRNA and protein synthesis by fluorescence spectroscopy at different concentrations of DNA template. Analysis of polysome distributions by atomic force microscopy, combined with a stochastic model of translation, revealed inefficient usage of ribosomes, consistent with the idea that translation initiation is a limiting step. This preliminary dataset was used to formulate hypotheses regarding possible mechanisms impeding robust gene expression. Next, we challenged these hypotheses by devising targeted experiments aimed to alleviate the current limitations of PUREfrex. We identified depletion of key initiation factors by translationally inactive mRNA as a possible inhibitory mechanism. This adverse process could partly be remedied by targeted mRNA degradation, whereas addition of more IFs and of the hrpA RNA helicase had no substantial effects. Moreover, depletion of tRNAs as peptidyl-tRNAs can become limiting in PUREfrex (but not in PURExpress), which can be alleviated by addition of peptidyl-tRNAhydrolase (PTH). We attempted to build a new model for PURE system dynamics integrating all experimental observations. Although a satisfying global fit can be obtained in specific conditions (with PTH), a unifying system's level model is still missing.

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Section: Attempts To Restore Transcription-translation Coupling Failementioning

confidence: 99%

Modelling cell-free RNA and protein synthesis with minimal systems

et al. 2019

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“…In a typical diluted in vitro environment, system stochasticity is often omitted due to the assumption that, under a well-stirred bulk environment, a low concentration of molecules and the random nature of their collisions can be eliminated, or at least greatly reduced [93]. However, the minimal CFPS system PURE hosts more than 900 reactions and involves the translation process [94], without considering the whole transcription process [95]. Therefore, this intrinsic randomness of the CFPS system cannot be completely ignored in micro-compartments, even under bulk conditions.…”

Section: Stochasticitymentioning

confidence: 99%

Cell-Free Protein Synthesis: Chassis toward the Minimal Cell

Zhu

Lou

2019

Cells

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The quest for a minimal cell not only sheds light on the fundamental principles of life but also brings great advances in related applied fields such as general biotechnology. Minimal cell projects came from the study of a plausible route to the origin of life. Later on, research extended and also referred to the construction of artificial cells, or even more broadly, as in vitro synthetic biology. The cell-free protein synthesis (CFPS) techniques harness the central cellular activity of transcription/translation in an open environment, providing the framework for multiple cellular processes assembling. Therefore, CFPS systems have become the first choice in the construction of the minimal cell. In this review, we focus on the recent advances in the quantitative analysis of CFPS and on its advantage for addressing the bottom-up assembly of a minimal cell and illustrate the importance of systemic chassis behavior, such as stochasticity under a compartmentalized micro-environment.

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“…Stochasticity due to the low concentration of molecules and the random nature of their collisions can be eliminated, or at least greatly reduced, via stirring . However, as indicated above, even with the most simplified CFPS system PURE, there would be more than 968 reactions in total, not even considering the whole transcription process involve both binding of RNA polymerase as well as the folding of mRNA . Therefore, this intrinsic randomness could not be completely ignored in microcompartments, perhaps even under bulk conditions.…”

Section: The Impact Of Stochasticitymentioning

confidence: 99%

Cell‐Free Protein Synthesis and Its Perspectives for Assembling Cells from the Bottom‐Up

Lou

Schwille

2019

Adv. Biosys.

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The underlying idea of synthetic biology is that biological reactions/modules/systems can be precisely engineered and controlled toward desired products. Numerous efforts in the past decades in deciphering the complexity of biological systems in vivo have led to a variety of tools for synthetic biology, especially based on recombinant DNA. However, one generic limitation of all living systems is that the vast majority of energy input is dedicated to maintain the system as a whole, rather than the small part of interest. Cell‐free synthetic biology is aiming at exactly this fundamental limitation, providing the next level of flexibility for engineering and designing biological systems in vitro. New technology has continuously inspired cell‐free biology and extended its applications, including gene circuits, spatiotemporally controlled pathways, coactivated catalysts systems, and rationally designed multienzyme pathways, in particular, minimal cell construction. In the context of this special issue, discussing work being carried out in the “MaxSynBio” consortium, the advances in characterizing stochasticity and dynamics of cell‐free protein synthesis within cell‐sized compartments, as well as the molecular crowding effect, are discussed. The organization of spatial heterogeneity is the key prerequisite for achieving hierarchy and stepwise assembly of minimal cells from the bottom‐up.

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Cell-Free Translation Is More Variable than Transcription

Cited by 42 publications

References 49 publications

Modelling cell-free RNA and protein synthesis with minimal systems

Modelling cell-free RNA and protein synthesis with minimal systems

Cell-Free Protein Synthesis: Chassis toward the Minimal Cell

Cell‐Free Protein Synthesis and Its Perspectives for Assembling Cells from the Bottom‐Up

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