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Engineering and testing RNA-circuits in cell-free systems

Lehr, François-Xavier (2021):
Engineering and testing RNA-circuits in cell-free systems. (Publisher's Version)
Darmstadt, Technische Universität,
DOI: 10.26083/tuprints-00015408,
[Ph.D. Thesis]

Abstract

RNA molecules lie at the heart of living organisms where they are associated with most of the cellular processes. They have recently emerged as one of the most promising elements for developing programmable genetic regulatory systems. RNA regulators have been shown to offer great advantages to harness the power of synthetic biology. Versatility of functions, predictability of design, and light metabolic cost have turned RNA-based devices into components of primordial importance for therapeutic, diagnostic and biotechnological applications. However, advanced tasks require the use of sequential logic circuits that embed many constituents in the same system. Combining RNA-parts into more complex circuits remains experimentally challenging and difficult to predict. Contrary to protein-based networks, little work has been performed regarding the integration of RNA components to multi-level regulated circuits. In the first part of this thesis, combinations of variety of small transcriptional activator RNAs (STARs) and toehold switches were built into highly effective AND-gates. To characterise the components and their dynamic range, an Escherichia coli (E. coli) cell-free transcription-translation (TX-TL) system dispensed via nanoliter droplets was used. Cell-free systems, which constitute an open environment, have removed many of the complexities linked to the traditional use of living cells and have led to exciting opportunities for the rational design of genetic circuits. A modelling framework based on ordinary differential equations (ODEs), where parameters were inferred via parallel tempering, was established to analyse the expression construct in a qualitative and quantitative manner. Based on this analysis, nine additional AND-gates were built and tested in vitro. The functionality of the gates was found to be highly dependent on the concentration of the activating RNA for either the STAR or the toehold switch. All gates were successfully implemented in vivo, displaying a dynamic range comparable to the level of protein circuits. Subsequent spacer screening experiment enabled isolation of a gate mutant with dynamic range up to 1087 fold change, paving the way towards multi-layered devices where tight OFF-stages are required for efficient computation. Expanding the repertoire of RNA regulatory parts with efficient inhibitors would complete the set of logic operations necessary for the building of dynamiccircuits, such as memory devices or oscillators. The TX-TL system was functionalized with pre-expressed dSpyCas9, a mutated version of Cas9 without endonuclease activity. Four functional small guide RNAs (sgRNAs) targeting the sfGFP reporter were engineered and characterized, all resulting in high repression efficiency. A three-inputs logic circuit containing toehold, STAR and sgRNA was successfully co-expressed, validating the orthogonality of NOT and AND gates based solely on RNA-based regulation. In order to minimize interactions which could arise from RNA-circuit of increasing complexity, the TX-TL system was functionalized with a second protein, the Csy4 endoribonuclease, which selectively binds and cuts a small RNA hairpin. Normalization of gene expression from various untranslated region contexts and enhanced processing of three-inputs small RNA operon were demonstrated via the use of Csy4. Finally, characterizing complex RNA-based circuits requires techniques that resolves dynamics. To overcome the batch-format limitations inherent to TX-TL systems, a microfluidic nanoliter-scaled reactor was implemented, enabling synthesis rates to stay constant over time. Dynamic control of RNA circuitry was demonstrated by modulating the concentration of ligands, reversing the gene state through the conformational change of riboswitches. This thesis shows the potential of a rapid prototyping approach for RNA circuit design in TX-TL systems combined with a predicting model framework. Taken together, the characterization of a variety of RNA-parts : activators, repressors, or controllers culminating into logic modules; and augmented cell-extracts; form a complete RNA-toolbox for cell-free systems. The leveraging of this unique prototyping platform will ultimately enable the engineering and the study of highly dynamical RNA-circuits in vitro.

Item Type: Ph.D. Thesis
Erschienen: 2021
Creators: Lehr, François-Xavier
Status: Publisher's Version
Title: Engineering and testing RNA-circuits in cell-free systems
Language: English
Abstract:

RNA molecules lie at the heart of living organisms where they are associated with most of the cellular processes. They have recently emerged as one of the most promising elements for developing programmable genetic regulatory systems. RNA regulators have been shown to offer great advantages to harness the power of synthetic biology. Versatility of functions, predictability of design, and light metabolic cost have turned RNA-based devices into components of primordial importance for therapeutic, diagnostic and biotechnological applications. However, advanced tasks require the use of sequential logic circuits that embed many constituents in the same system. Combining RNA-parts into more complex circuits remains experimentally challenging and difficult to predict. Contrary to protein-based networks, little work has been performed regarding the integration of RNA components to multi-level regulated circuits. In the first part of this thesis, combinations of variety of small transcriptional activator RNAs (STARs) and toehold switches were built into highly effective AND-gates. To characterise the components and their dynamic range, an Escherichia coli (E. coli) cell-free transcription-translation (TX-TL) system dispensed via nanoliter droplets was used. Cell-free systems, which constitute an open environment, have removed many of the complexities linked to the traditional use of living cells and have led to exciting opportunities for the rational design of genetic circuits. A modelling framework based on ordinary differential equations (ODEs), where parameters were inferred via parallel tempering, was established to analyse the expression construct in a qualitative and quantitative manner. Based on this analysis, nine additional AND-gates were built and tested in vitro. The functionality of the gates was found to be highly dependent on the concentration of the activating RNA for either the STAR or the toehold switch. All gates were successfully implemented in vivo, displaying a dynamic range comparable to the level of protein circuits. Subsequent spacer screening experiment enabled isolation of a gate mutant with dynamic range up to 1087 fold change, paving the way towards multi-layered devices where tight OFF-stages are required for efficient computation. Expanding the repertoire of RNA regulatory parts with efficient inhibitors would complete the set of logic operations necessary for the building of dynamiccircuits, such as memory devices or oscillators. The TX-TL system was functionalized with pre-expressed dSpyCas9, a mutated version of Cas9 without endonuclease activity. Four functional small guide RNAs (sgRNAs) targeting the sfGFP reporter were engineered and characterized, all resulting in high repression efficiency. A three-inputs logic circuit containing toehold, STAR and sgRNA was successfully co-expressed, validating the orthogonality of NOT and AND gates based solely on RNA-based regulation. In order to minimize interactions which could arise from RNA-circuit of increasing complexity, the TX-TL system was functionalized with a second protein, the Csy4 endoribonuclease, which selectively binds and cuts a small RNA hairpin. Normalization of gene expression from various untranslated region contexts and enhanced processing of three-inputs small RNA operon were demonstrated via the use of Csy4. Finally, characterizing complex RNA-based circuits requires techniques that resolves dynamics. To overcome the batch-format limitations inherent to TX-TL systems, a microfluidic nanoliter-scaled reactor was implemented, enabling synthesis rates to stay constant over time. Dynamic control of RNA circuitry was demonstrated by modulating the concentration of ligands, reversing the gene state through the conformational change of riboswitches. This thesis shows the potential of a rapid prototyping approach for RNA circuit design in TX-TL systems combined with a predicting model framework. Taken together, the characterization of a variety of RNA-parts : activators, repressors, or controllers culminating into logic modules; and augmented cell-extracts; form a complete RNA-toolbox for cell-free systems. The leveraging of this unique prototyping platform will ultimately enable the engineering and the study of highly dynamical RNA-circuits in vitro.

Place of Publication: Darmstadt
Collation: xxxi, 124 Seiten
Divisions: 18 Department of Electrical Engineering and Information Technology
18 Department of Electrical Engineering and Information Technology > Institute for Telecommunications > Bioinspired Communication Systems
18 Department of Electrical Engineering and Information Technology > Institute for Telecommunications
Date Deposited: 12 Mar 2021 14:23
DOI: 10.26083/tuprints-00015408
Official URL: https://tuprints.ulb.tu-darmstadt.de/15408
URN: urn:nbn:de:tuda-tuprints-154087
Referees: Koeppl, Prof. Dr. Heinz ; Goeringer, Prof. Dr. H. Ulrich ; Beisel, Prof. Chase ; Stein, Prof. Dr. Viktor
Refereed / Verteidigung / mdl. Prüfung: 30 April 2020
Alternative Abstract:
Alternative abstract Language

RNA-Moleküle bilden ein Kernstück lebender Organismen, wo sie mit den meisten zellulären Prozessen in Verbindung gebracht werden. Sie sind in jüngerer Zeit hervorgetreten als eines der vielversprechendsten Elemente für die Entwicklung programmierbarer genetischer Regulationssysteme. RNA-Regulatoren bieten große Vorteile dabei, die Kraft der synthetischen Biologie zu nutzen. Durch die Vielseitigkeit ihrer Funktionen, der Vorhersagbarkeit beim Design und ihrer geringen Stoffwechselkosten sind RNA-basierte Elemente von grundlegender Bedeutung für therapeutische, diagnostische und biotechnologische Anwendungen. Fortgeschrittene Aufgaben erfordern jedoch die Verwendung von sequentiellen Logikschaltungen, die viele Bestandteile in dasselbe System einbetten. Das Kombinieren von RNA-Komponenten zu komplexeren Schaltkreisen bleibt experimentell herausfordernd und schwierig vorherzusagen. Im Gegensatz zu proteinbasierten Netzwerken wurden nur wenige Arbeiten zur Integration von RNA-Komponenten in mehrstufig geregelte Schaltkreise durchgeführt. Im ersten Teil dieser Arbeit wurden Kombinationen verschiedener kleiner Transkriptionsaktivator-RNAs, small transcriptional activator RNAs (STARs), und Toehold-Schalter in hochwirksame AND-Gatter eingebaut. Zur Charakterisierung der Komponenten und ihres Dynamikbereichs wurde ein zellfreies Escherichia coli (E. coli) -Transkriptions-Translations-System (TX-TL) verwendet, das über Nanolitertröpfchen verteilt wurde. Zellfreie Systeme, die eine offene Umgebung darstellen, haben viele der Komplexitäten, die mit der traditionellen Verwendung lebender Zellen verbunden sind, beseitigt und aufregende Möglichkeiten für die rationale Gestaltung genetischer Schaltkreise eröffnet. Zur qualitativen und quantitativen Analyse des Expressionskonstrukts wurde ein auf gewöhnlichen Differentialgleichungen, ordinary differential equations (ODEs), basierendes Modellierungsframework erstellt, dessen Parameter durch parallel tempering abgeleitet wurden. Basierend auf dieser Analyse wurden neun zusätzliche AND-Gatter gebaut und in vitro getestet. Es wurde festgestellt, dass die Funktionalität der Gatter stark von der Konzentration der aktivierenden RNA für den STAR- beziehungsweise den Toehold-Schalter abhängt. Alle Gatter wurden erfolgreich in vivo implementiert und wiesen einen Dynamikbereich auf, der mit dem von Proteinkreisläufen vergleichbar ist. Ein anschließendes Spacer-Screening-Experiment ermöglichte die Isolierung einer Gatter-Mutante mit einem Dynamikbereich von bis zu 1087-facher Veränderung, womit der Weg geebnet wurde für mehrschichtige Bauelemente, bei denen enge OFF-Level für eine effiziente Schaltung erforderlich sind. Die Erweiterung des Repertoires der RNA-Regulatoren um wirksame Inhibitoren würde die für den Aufbau dynamischer Schaltkreise, wie Speicherbausteine oder Oszillatoren, erforderlichen logischen Operationen vervollständigen. Das TX-TL-System wurde mit vorexprimiertem dSpyCas9, einer mutierten Version von Cas9 ohne Endonukleaseaktivität, funktionalisiert. Vier funktionelle Small-Guide-RNAs (sgRNAs), die auf den sfGFP-Reporter abzielen, wurden konstruiert und charakterisiert und wiesen eine hohe Repressionseffizienz auf. Eine Logikschaltung mit drei Eingängen, die Toehold, STAR und sgRNA enthielt, wurde erfolgreich co-exprimiert und validierte die Orthogonalität von NOT- und AND-Gattern ausschließlich auf der Grundlage der RNA-basierten Regulation. Um Wechselwirkungen zu minimieren, die durch einen immer komplexer werdenden RNA-Schaltkreis entstehen könnten, wurde das TX-TL-System mit einem zweiten Protein, der Csy4-Endoribonuklease, funktionalisiert, welches selektiv eine kleine RNA-Haarnadel bindet und schneidet. Die Normierung der Genexpression verschiedener nicht translatierter Regionskontexte und die verbesserte Verarbeitung von kleinen RNA-Operons mit drei Eingängen wurde mithilfe von Csy4 demonstriert. Zuletzt erfordert die Charakterisierung komplexer RNA-basierter Schaltkreise Techniken um Dynamiken zu erfassen. Um die mit TX-TL-Systemen verbundenen Einschränkungen des Batch-Formats zu überwinden, wurde ein mikrofluidischer Nanoliter-Reaktor implementiert, der es ermöglicht, die Syntheseraten über die Zeit konstant zu halten. Die dynamische Kontrolle der RNA-Schaltkreise wurde durch Modulation der Ligandenkonzentration und Umkehrung des Genzustands durch Konformationsänderung von Riboschaltern demonstriert. Diese Arbeit zeigt das Potenzial eines Rapid-Prototyping-Ansatzes für das Design von RNA-Schaltkreisen in TX-TL-Systemen in Kombination mit einem Modellierungsframework. Zusammengefasst bilden die Charakterisierung einer Vielzahl von RNA-Teilen wie Aktivatoren, Repressoren oder Controllern, die in Logikmodulen gipfeln, sowie funktionserweiterte Zellextrakte, eine komplette RNA-Toolbox für zellfreie Systeme. Die Nutzung dieser einzigartigen Prototyping- Plattform wird es letztendlich ermöglichen, hochdynamische RNA-Schaltkreise in vitro zu konstruieren und zu untersuchen.

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