DNA-functionalized hydrogels for confined membrane-free in vitro transcription/translation

Thiele, Julian; Ma, Yujie; Foschepoth, David; Hansen, Maike M. K.; Steffen, Christian; Heus, Hans A.; Huck, Wilhelm T. S.

doi:10.1039/c3lc51427g

Cited by 47 publications

(61 citation statements)

References 37 publications

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“…(b) Fabrication of hybrid DNA‐hyaluronic acid hydrogel microparticles by functionalization of thiol‐modified hyaluronic acid with phosphoramidite DNA to give HA‐S‐DNA and its subsequent crosslinking with PEGDA and labeling using thiol‐reactive Alexa 555 maleimide in a microfluidic flow focusing device. From Thiele et al (2014), DNA‐functionalized hydrogels for confined membrane‐free in vitro transcription/translation, Lab Chip , 14(15), 2651–2656, https://doi.org/10.1039/C3LC51427G, with permission of the Royal Society of Chemistry (Great Britain). (c) Design of DNA brush capable of programmable on‐chip DNA to realize artificial cells.…”

Section: Cfps: Latest Platforms and Applications In Biotechnology Andmentioning

confidence: 99%

Cell‐free protein synthesis: The transition from batch reactions to minimal cells and microfluidic devices

Ayoubi‐Joshaghani

Dianat‐Moghadam

Seidi

et al. 2020

Biotech & Bioengineering

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Thanks to the synthetic biology, the laborious and restrictive procedure for producing a target protein in living microorganisms by biotechnological approaches can now experience a robust, pliant yet efficient alternative. The new system combined with lab‐on‐chip microfluidic devices and nanotechnology offers a tremendous potential envisioning novel cell‐free formats such as DNA brushes, hydrogels, vesicular particles, droplets, as well as solid surfaces. Acting as robust microreactors/microcompartments/minimal cells, the new platforms can be tuned to perform various tasks in a parallel and integrated manner encompassing gene expression, protein synthesis, purification, detection, and finally enabling cell‐cell signaling to bring a collective cell behavior, such as directing differentiation process, characteristics of higher order entities, and beyond. In this review, we issue an update on recent cell‐free protein synthesis (CFPS) formats. Furthermore, the latest advances and applications of CFPS for synthetic biology and biotechnology are highlighted. In the end, contemporary challenges and future opportunities of CFPS systems are discussed.

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Section: Cfps: Latest Platforms and Applications In Biotechnology Andmentioning

confidence: 99%

Cell‐free protein synthesis: The transition from batch reactions to minimal cells and microfluidic devices

Ayoubi‐Joshaghani

Dianat‐Moghadam

Seidi

et al. 2020

Biotech & Bioengineering

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“…In this context, membrane-free compartments such as chip-based gene expression systems [6] , bead-based systems [7] , coacervates [8] or hydrogels [9] have been more successfully utilized to employ systems dynamics involving diffusible nucleic acid signals. Among other applications, such systems were used to engineer biochemical reaction networks that exhibit a variety of spatial patterns and wave-like dynamics [7, 10] , or to mimic predatory behavior [11] .…”

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confidence: 99%

“…The experimental workflow (Figure 1a, SI Section 1) was adapted from Thiele et al [9] and consists of chemical functionalization of the hydrogel (step 1) for subsequent covalent modification with PCR amplified DNA (step 2). The resulting DNA hydrogel is first encapsulated in emulsion droplets of diameter d ≈ 30 µm employing microfluidic flow focusing and afterwards gelled (step 3).…”

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confidence: 99%

Artificial Gel‐Based Organelles for Spatial Organization of Cell‐Free Gene Expression Reactions

Aufinger

Simmel

2018

Angew Chem Int Ed

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In biological cells, chemical processes often occur inside specialized subcellular compartments or organelles. For instance, in eukaryotes mRNA is transcribed and processed inside the nucleus, exported to the endoplasmic reticulum, and translated into the encoded protein. Inspired by this high degree of intracellular organization, we here develop gel-based artificial organelles that enable sequence-specific and programmable localization of cell-free transcription and translation reactions inside an artificial cellular system. To this end, we utilize agarose microgels covalently modified with DNA templates coding for various functions and encapsulate them into emulsion droplets. We show that RNA signals transcribed from transcription organelles can be specifically targeted to capture organelles via hybridization to the corresponding DNA addresses. We also demonstrate that mRNA molecules, produced from transcription organelles and controlled by toehold switch riboregulators, are only translated in translation organelles containing their cognate DNA triggers. Spatial confinement of transcription and translation in separate organelles is thus superficially similar to gene expression in eukaryotic cells. Combining communicating gel spheres with specialized functions opens up new possibilities for programming artificial cellular systems at the organelle level.

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“…In diesem Zusammenhang wurden zuletzt membranfreie Kompartimente wie chipbasierte Genexpressionssysteme, [9] Mikrokügelchen, [10] Koazervate [11] oder Hydrogele [12] fürd ie Realisierung dynamischer Systeme mit diffundierenden Nukleinsäuresignalen verwendet. Zusätzlich zu anderen Anwendungen wurden mit solchen Systemen auch biochemische Reaktionsnetzwerke realisiert, die räumliche Muster oder wellenartige Dynamik generieren [10,13] oder auch Jäger-Beute-Dynamik imitieren konnten.…”

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Künstliche, gelbasierte Organellen für die räumliche Organisation von zellfreien Genexpressionsreaktionen

Aufinger

Simmel

2018

Angewandte Chemie

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Gelbasierte,k ünstliche Organellen wurden entwickelt, die die sequenzspezifische und programmierbare Lokalisation zellfreier Transkriptions-und Translationsreaktionen innerhalb eines künstlichen zellulären Systems ermçglichen. Dazu wurden Agarose-Mikrogele genutzt, die kovalent mit DNA-Templaten modifiziert wurden, die fürverschiedene Funktionen codieren, und diese wiederum in Emulsionstrçpfchen eingeschlossen. VonT ranskriptionsorganellent ranskribierte RNA-Signale kçnnen spezifisch,d urchH ybridisierung mit entsprechenden DNA-Adressmolekülen, von Zielorganellen eingefangen werden. mRNA-Moleküle mit Toehold-Switch-Riboregulatoren, die von Transkriptionsorganellen produziert werden, werden nur in Translationsorganellen exprimiert, die den entsprechenden DNA-Trigger fürd en Riboregulator enthalten. Die räumlicheB eschränkung von Transkription und Translation auf getrennte Organellen ähnelt damit der Genexpression in eukaryotischen Zellen. Die Kombination kommunizierender Gelkügelchen mit spezialisierten Funktionen erçffnet die Mçglichkeit der Programmierung künstlicher zellulärer Systeme auf Organell-Ebene.

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DNA-functionalized hydrogels for confined membrane-free in vitro transcription/translation

Cited by 47 publications

References 37 publications

Cell‐free protein synthesis: The transition from batch reactions to minimal cells and microfluidic devices

Cell‐free protein synthesis: The transition from batch reactions to minimal cells and microfluidic devices

Artificial Gel‐Based Organelles for Spatial Organization of Cell‐Free Gene Expression Reactions

Künstliche, gelbasierte Organellen für die räumliche Organisation von zellfreien Genexpressionsreaktionen

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