Programmable on-chip DNA compartments as artificial cells

Karzbrun, Eyal; Tayar, Alexandra M.; Noireaux, Vincent; Bar‐Ziv, Roy

doi:10.1126/science.1255550

Cited by 247 publications

(290 citation statements)

References 27 publications

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“…Recently cell-free systems have begun to gain a foothold as viable platforms for synthetic biology [8][9][10] . The appeal of such approaches is that they free synthetic biology from the resource sharing and 'extrinsic noise' that affects reaction dynamics in living cells.…”

Section: Introductionmentioning

confidence: 99%

Sealable Femtoliter Chamber Arrays for Cell-free Biology

Norred

Caveney

Retterer

et al. 2015

JoVE

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Cell-free systems provide a flexible platform for probing specific networks of biological reactions isolated from the complex resource sharing (e.g., global gene expression, cell division) encountered within living cells. However, such systems, used in conventional macro-scale bulk reactors, often fail to exhibit the dynamic behaviors and efficiencies characteristic of their living micro-scale counterparts. Understanding the impact of internal cell structure and scale on reaction dynamics is crucial to understanding complex gene networks. Here we report a microfabricated device that confines cell-free reactions in cellular scale volumes while allowing flexible characterization of the enclosed molecular system. This multilayered poly(dimethylsiloxane) (PDMS) device contains femtoliter-scale reaction chambers on an elastomeric membrane which can be actuated (open and closed). When actuated, the chambers confine Cell-Free Protein Synthesis (CFPS) reactions expressing a fluorescent protein, allowing for the visualization of the reaction kinetics over time using time-lapse fluorescent microscopy. Here we demonstrate how this device may be used to measure the noise structure of CFPS reactions in a manner that is directly analogous to those used to characterize cellular systems, thereby enabling the use of noise biology techniques used in cellular systems to characterize CFPS gene circuits and their interactions with the cell-free environment.

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Section: Introductionmentioning

confidence: 99%

Sealable Femtoliter Chamber Arrays for Cell-free Biology

Norred

Caveney

Retterer

et al. 2015

JoVE

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“…For vertebrates, negatively charged polysaccharide hyaluronic acids play a vital role in the organization of pericellular matrix [2,3]. Double-stranded DNA brushes on a biochip [4] at a cell-like density (∼ 10 4 kb/µm 3 ) have been developed as a platform to study cell-free gene expression [5]. Due to the negative charges along the backbone, this synthetic system behaves like a well-defined strong polyelectrolyte brush [5], the height of which can be varied by modulating the ionic strength of monovalent salt (NaCl) solution and the grafting density [6].…”

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confidence: 99%

Heterogeneous Morphology and Dynamics of Polyelectrolyte Brush Condensates in Trivalent Counterion Solution

2017

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Recent experiments have shown that trivalent ion, spermidine 3+ , can provoke lateral microphase segregation in DNA brushes. Using molecular simulations and simple theoretical arguments, we explore the effects of trivalent counterions on polyelectrolyte brushes. At a proper range of grafting density, polymer size, and ion concentration, the brush polymers collapse heterogeneously into octopus-like surface micelles. Remarkably, the heterogeneity in brush morphology is maximized and the relaxation dynamics of chain and condensed ion are the slowest at the 1:3 stoichiometric concentration of trivalent ions to polyelectrolyte charge. A further increase of trivalent ion concentration conducive to a charge inversion elicits modest reswelling and homogenizes the morphology of brush condensate. Our study provides a new insight into the origin of the diversity in DNA organization in cell nuclei as well as the ion-dependent morphological variation in polyelectrolyte brush layer of biological membranes.Polyelectrolyte brushes are ubiquitous in biological systems. As the main component of outer membrane in Gram-negative bacteria, lipopolysaccharides, consisting of charged O-polysaccharides side chains, form a brush layer and mediate the interaction of bacteria with their environment [1]. For vertebrates, negatively charged polysaccharide hyaluronic acids play a vital role in the organization of pericellular matrix [2,3]. Double-stranded DNA brushes on a biochip [4] at a cell-like density (∼ 10 4 kb/µm 3 ) have been developed as a platform to study cell-free gene expression [5]. Due to the negative charges along the backbone, this synthetic system behaves like a well-defined strong polyelectrolyte brush [5], the height of which can be varied by modulating the ionic strength of monovalent salt (NaCl) solution and the grafting density [6]. Recently, it has been reported that trivalent counterions, spermidine 3+ (Spd 3+ ), can induce a collapse transition of DNA brush into fractal-like dendritic macroscopic domains [7]. The heterogeneous morphology of DNA condensate points to polyamine-mediated regulation of local DNA configuration and gene expression [8,9], underscoring the importance of understanding the effects of multivalent counterions on polyelectrolyte brush [10,11]. Compared with uncharged polymer brushes in good or poor solvent, the electrostatic interaction and osmotic pressure, which are readily controlled by the salt concentration and pH, yield additional flexibilities in manipulating polyelectrolyte brushes, and thus holding promise for its wide applications [12,13].Conventional theoretical approaches, successful in explaining the behaviors of polyelectrolytes in monovalent salt solutions [11,14,15], are of limited use, particularly, when multivalent counterions are at work. For example, the solution to the mean-field PoissonBoltzmann theory [16] fails to account for phenomena such as ion condensation, charge over-compensation, and collapse of like-charged polymers [17][18][19][20]. A prediction from a nonlocal den...

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“…Very recently, two-dimensional DNA compartments in silicon were generated [68]. In these compartments protein expression cycles can be auto-regulated using interconnected compartments containing different sets of DNA.…”

Section: Cell-like Systemsmentioning

confidence: 99%

Cell-Free Synthesis of Macromolecular Complexes

Botte

Deniaud

Schaffitzel

2016

Advanced Technologies for Protein Complex Production and Characterization

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General rightsThis document is made available in accordance with publisher policies. Please cite only the published version using the reference above. Full terms of use are available: http://www.bristol.ac.uk/pure/about/ebr-terms ABSTRACTCell-free protein synthesis based on E. coli cell extracts has been described for the first time more than fifty years ago. To date, cell-free synthesis is widely used for the preparation of toxic proteins, for studies of the translation process and its regulation as well as for the incorporation of artificial or labelled amino acids into a polypeptide chain. Many efforts have been directed towards establishing cell-free expression as a standard method for gene expression, with limited success. In this chapter we will describe the state-of-theart of cell-free expression, extract preparation methods and recent examples for successful applications of cell-free synthesis of macromolecular complexes.3 1/ Introduction -Motivation and challengesThe capacity of cell extracts to synthesize proteins has been shown in the fifties of the last century [1,2], several years before the identification of ribosomes as proteinsynthesizing machines [3]. The cell-free extract was based on the classical S30 fraction obtained by a 30,000 x g centrifugation step at 4°C for 1 hour. Initially, endogenous mRNA was used for in vitro translation [4]. Subsequently, Nirenberg and Matthaei developed a protocol to degrade endogenous messenger RNA present in the cell extract and to add exogenous mRNA [5,6]. The first cell-free protein synthesis (CFPS) from DNA, using a socalled coupled transcription-translation system was developed in the late sixties by the group of Zubay [7]. They used their coupled transcription-translation system to study the regulation of gene expression by the E. coli lactose operon. Most cell-free extract preparation and in vitro translation protocols are based on this protocol [8,9].Significant improvements with respect to protein yields were achieved in the late eighties, in particular by the group of Spirin, which established the use of phage-specific RNA polymerases, SP6 [10] or T7 RNA polymerases [8]. Using these polymerases a high level of a specific mRNA during the in vitro transcription-translation reaction can be achieved and maintained. Importantly, the Spirin laboratory described the first 'continuous' in vitro translation system. It allows for a continuous exchange of small molecules between a 'feeding compartment' providing energy and substrates (amino acids) for the translation reaction and a 'reaction compartment' from which inhibitory reaction products are removed by dialysis [10,11]. In a continuous set-up, the in vitro translation reaction can continue for several hours or even days, compared to 40-60 minutes using the classical reaction set-up.This allows obtaining significantly increased yields: for instance 6 mg chloramphenicol Cell-free expression has thus several very attractive applications, related to the expression of toxic proteins, rapid production of small q...

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Programmable on-chip DNA compartments as artificial cells

Cited by 247 publications

References 27 publications

Sealable Femtoliter Chamber Arrays for Cell-free Biology

Sealable Femtoliter Chamber Arrays for Cell-free Biology

Heterogeneous Morphology and Dynamics of Polyelectrolyte Brush Condensates in Trivalent Counterion Solution

Cell-Free Synthesis of Macromolecular Complexes

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