Towards a synthetic metabolism: exploitation of synthetic and natural nucleotide synthesis pathways for in vitro gene expression

Contains fulltext : 175865.pdf (publisher's version ) (Open Access)Radboud University, 11 september 2017Promotores : Huck, W.T.S., Hest, J.C.M. van Co-promotor : Heus, H.A.ii, 130 p

Macromolecular Crowding in the Cytosol: Underappreciated or Overestimated?

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In vitro synthesis of 32 translation-factor proteins from a single template reveals impaired ribosomal processivity

The Protein synthesis Using Recombinant Elements (PURE) system enables transcription and translation of a DNA template from purified components. Therefore, the PURE system-catalyzed generation of RNAs and proteins constituting the PURE system itself represents a major challenge toward a self-replicating minimal cell. In this work, we show that all translation factors (except elongation factor Tu) and 20 aminoacyl-tRNA synthetases can be expressed in the PURE system from a single plasmid encoding 32 proteins in 30 cistrons. Cell-free synthesis of all 32 proteins is confirmed by quantitative mass spectrometry-based proteomic analysis using isotopically labeled amino acids. We find that a significant fraction of the gene products consists of proteins missing their C-terminal ends. The per-codon processivity loss that we measure lies between 1.3 x 10(-3) and 13.2 x 10(-3), depending on the expression conditions, the version of the PURE system, and the coding sequence. These values are 5 to 50 times higher than those measured in vivo in E. coli. With such an impaired processivity, a considerable fraction of the biosynthesis capacity of the PURE system is wasted, posing an unforeseen challenge toward the development of a self-regenerating PURE system

Datenschutz - aktuelle Fragen und Antworten : Atzelsberger Gespräche 2014

Nicht erst, aber vor allem infolge der Enthüllungen des ehemaligen US-Geheimdienstmitarbeiters Edward Snowden ist „Datenschutz“ zu einem großen Thema unserer Zeit geworden. Die 33. Atzelsberger Gespräche der Dr. Alfred-Vinzl-Stiftung an der Friedrich-Alexander-Universität Erlangen-Nürnberg widmeten sich ihm in Teilaspekten, indem die Referenten und übrigen Gesprächsteilnehmer zur komplizierten Gesamtproblematik in unterschiedlicher Weise Fragen aufwarfen, Antworten gaben und eine anregende Diskussion belebten. Der vorliegende Band enthält die vier Vorträge in überarbeiteter Fassung. Der Freiburger Historiker Josef Foschepoth fragt in seinem Beitrag auf der Grundlage seines bereits in vierter Auflage vorliegenden Buches „Überwachtes Deutschland“ nach dem Zusammenhang von „Verfassung und Wirklichkeit“ am Beispiel der „Überwachung des Post- und Fernmeldeverkehrs in der Geschichte der Bundesrepublik Deutschland“. Um „Völker- und menschenrechtliche Anforderungen an Informations-beschaffung und Datenüberwachung durch ausländische Geheimdienste“ geht es dem Erlanger Völker- und Öffentlichrechtler Markus Krajewski, während der Nürnberger Steuerrechtler Roland Ismer nicht nur vor dem Hintergrund des Ankaufs in der Schweiz illegal kopierter „Steuer CDs“ das Thema „Datenschutz im Steuerrecht“ behandelt. Schließlich stellt Thomas Kranig, der Präsident des Bayerischen Landesamts für Daten-schutzaufsicht die Frage „Datenschutz und Datensicherheit – mission impossible?“ und gibt zugleich Einblicke in seine in Ansbach ansässige Behörde

In vitro synthesis of 32 translation-factor proteins from a single template reveals impaired ribosomal processivity

The Protein synthesis Using Recombinant Elements (PURE) system enables transcription and translation of a DNA template from purified components. Therefore, the PURE system-catalyzed generation of RNAs and proteins constituting the PURE system itself represents a major challenge toward a self-replicating minimal cell. In this work, we show that all translation factors (except elongation factor Tu) and 20 aminoacyl-tRNA synthetases can be expressed in the PURE system from a single plasmid encoding 32 proteins in 30 cistrons. Cell-free synthesis of all 32 proteins is confirmed by quantitative mass spectrometry-based proteomic analysis using isotopically labeled amino acids. We find that a significant fraction of the gene products consists of proteins missing their C-terminal ends. The per-codon processivity loss that we measure lies between 1.3 × 10–3 and 13.2 × 10–3, depending on the expression conditions, the version of the PURE system, and the coding sequence. These values are 5 to 50 times higher than those measured in vivo in E. coli. With such an impaired processivity, a considerable fraction of the biosynthesis capacity of the PURE system is wasted, posing an unforeseen challenge toward the development of a self-regenerating PURE system.</p

Genetically controlled membrane synthesis in liposomes

Lipid membranes, nucleic acids, proteins, and metabolism are essential for modern cellular life. Synthetic systems emulating the fundamental properties of living cells must therefore be built upon these functional elements. In this work, phospholipid-producing enzymes encoded in a synthetic minigenome are cell-free expressed within liposome compartments. The de novo synthesized metabolic pathway converts precursors into a variety of lipids, including the constituents of the parental liposome. Balanced production of phosphatidylethanolamine and phosphatidylglycerol is realized, owing to transcriptional regulation of the activity of specific genes combined with a metabolic feedback mechanism. Fluorescence-based methods are developed to image the synthesis and membrane incorporation of phosphatidylserine at the single liposome level. Our results provide experimental evidence for DNA-programmed membrane synthesis in a minimal cell model. Strategies are discussed to alleviate current limitations toward effective liposome growth and self-reproduction.BN/Christophe Danelon La

Cell-Like Nanostructured Environments Alter Diffusion and Reaction Kinetics in Cell-Free Gene Expression

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

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

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