Figuren 1, 2, 3, 4, 6, 8 und den Tabellen 2, 3, 10, 15, 16 sind aus Lit. 29 modifiziert. Diese Arbeit wurde von der National Science Foundation (NSF gewähren CCF-1.117.143 und NSF-CCF 1.162.141 auf GS). Y.-JC wurde vom taiwanesischen Regierung Stipendien unterstützt. SDR wurde von der National Science Foundation Graduate Research Fellowship Program (GRFP) unterstützt.

<ol>
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The authors declare no competing financial interests.

Dieser Artikel beschreibt ein Verfahren zum Ableiten ndsDNA Gates aus hochreiner Plasmid-DNA. Darüber hinaus wird ein Protokoll zur Charakterisierung Gate Leistung durch einen Fluoreszenz-Assay-Kinetik dargestellt. Experimentelle Daten zeigen, dass das Plasmid stammSystem trifft sein synthetisches Gegenstück, selbst wenn das synthetische System ist aus Strängen mit Polyacrylamid-Gelelektrophorese (PAGE) gereinigt, montiert. Wahrscheinlich ist die verbesserte Leistung von Plasmid stammende Gates vor allem auf die hohe Reinheit der biologischen DNA. Synthetische DNA enthält eine Vielzahl von Fehlern, insbesondere Deletionen, die Oligonukleotide mit einer Länge von n-1, und solche Nebenprodukte entstehen, werden in der Regel nicht vollständig PAGE oder Hochleistungs-Flüssigkeits-Chromatographie (HPLC) Reinigungsverfahren entfernt. Ähnliche Verbesserungen bei den hier berichteten wurden in einer früheren Studie von einer katalysierten Haarnadel-Verstärker, der aus biologischen Quellen 21 abgeleitete DNA verwendet beobachtet. <p class = &quot;jove_content&quot;&gt; Aber auch die Verwendung von Plasmid-abgeleitete Gatter können nicht vollständig beseitigen Fehler in der Gate Leistung, für die es mindestens zwei Gründe: Erstens über Verdauung oder Mangel an Schnittgenauigkeit können die Gates mit zu vielen führen Einschnitte oder Kerben in den falschen Positionen. In jedem Fall sind Gates eher in unerwünschter Reaktionen teilzunehmen. Solche Probleme können durch eine Optimierung der Menge des verwendeten Enzyms (siehe Abbildung 4) gelockert werden. Zweitens, in diesen Experimenten am Ein- und Hilfsstränge wurden synthetische DNA und enthielt Deletionen und Mutationen bei. Prinzipiell sind alle Einzelstrang Antriebs- und Hilfs Stränge könnte auch aus Phagemid-DNA durch eine Nicking-Enzym-Verdauung des vorcodierten m13 Virusgenom 26 erhalten werden. Vielleicht die Leistungsfähigkeit der Schaltung kann ferner unter Verwendung von ssDNA aus dem Bakteriengenom abgeleitet verbessert werden. Während die Verwendung von Plasmid-abgeleitete Gattern wurde gefunden, daß die Schaltungsleistung zu verbessern, eine Analyseder Kosten und Bearbeitungszeiten ergeben, dass, während die Herstellung von Plasmid stammende Gates etwas günstiger ist (Tabelle 15), dauert es 2-3 mal längere Verarbeitungszeit im Vergleich zur Montage und Reinigung von Gattern aus kommerziell synthetisiert Oligos (Tabelle 16). Die primären Kosten von Plasmid stammende Gates Gensynthese und die Verwendung von Restriktionsenzymen. 300 pmol von Gattern (ausreichend für 15 Reaktionen bei 30 nM), die geschätzten Kosten für die Join-Gatter ist ungefähr $ 170 und $ 200 für Gabel Tore, die Kostendifferenz wobei aufgrund der Verwendung von unterschiedlichen Nicking-Enzyme. Im Gegensatz dazu ist die chemische Synthese der Stränge für die gleiche Gate kostet rund 260 $ einschließlich einer PAGE Reinigungsgebühr. Die primären Zeitkosten für Plasmid stamm Toren ist in der Klonierung, die, genau wie die DNA-Synthese, kann zu einer Gensynthese Unternehmen ausgelagert werden. Haben jedoch nach der Montage Plasmid stamm Gatter den Vorteil, dass der Host-Plasmide können leicht zu einem replizierendennd in Form von bakteriellen Glycerinstamm gespeichert werden. Dadurch ist es möglich, um die Gates mehrfach wieder verwenden. Wir freuen uns, könnte die verbesserte Leistung von Plasmid stamm Toren einen wesentlich größeren Dynamikbereich als Verhaltensweisen sind experimentell bisher mit DNA CRNs gezeigt zu ermöglichen. Zum Beispiel schlug den letzten theoretischen Arbeit 47,48, dass selbstorganisierte räumliche Muster auf der Makroskala kann mit DNA CRNs durch eine Reaktionsdiffusionsmechanismus realisiert werden. Die hier vorgestellte Methode bietet eine praktikable Weg zum Aufbau der zugrunde liegenden molekularen Komponenten für solche Selbst Strukturieren DNA Materialien. Obwohl anspruchsvoll, die Entwicklung Makromaßstab Morphologien in einer programmierbaren Weise würde erhebliche Auswirkungen in Bereichen wie Biomaterialforschung an der regenerativen Medizin haben.

Die Vorhersehbarkeit der Watson-Crick-Basenpaarung konnten dynamischen DNA-Nanotechnologie als eine programmierbare Weg zu molekularen Geräte mit dynamischen Eigenschaften 1,2 entwerfen entstehen. Insbesondere DNA Strangverdrängung - eine programmierbare, wettbewerbsfähige Hybridisierungsreaktion - hat sich als leistungsfähigen Mechanismus für das Engineering dynamischer DNA Systeme sein. In einem DNA-Strang Verdrängungsreaktion, verdrängt ein eingehender Oligonukleotid eine zuvor gebunden &quot;output&quot; Strang aus einem komplementären Bindungspartner. Mehrere solche Reaktionen können zusammen in mehrstufigen Reaktorkaskaden mit einem hohen Grad an Kontrolle über die Reihenfolge und das Timing der einzelnen Reaktionsschritte 3 verkettet werden. DNA-Strangverdrängungs Kaskaden verwendet worden, um digitale und analoge Schaltungen Molekular 4-7, umschaltbar Nanostrukturen 8-10 autonome molekulare Motoren 11-15, und nicht-kovalente katalytischer Verstärker 13,16-21 erstellen. Außerdem DNA Geräte mit Strangverdrängungsreaktionen können simuliert und für vielfältige Anwendungen unter Verwendung von computergestützten Design-Software 22-24 ausgelegt sein. Derzeit ist chemisch synthetisierte DNA als Hauptmaterial für die DNA-Nanotechnologie. Jedoch Fehler in der DNA-Syntheseverfahren und die so erhaltenen unvollkommen Oligonukleotiden wird angenommen, dass die Leistung von dynamischen DNA von Geräten durch fehlerhafte Nebenreaktionen zu begrenzen. Zum Beispiel &quot;Leck&quot; Reaktionen können in der Freisetzung eines Ausgangs Oligonucleotid selbst in der Abwesenheit eines Reaktionstrigger führen. Solche Effekte werden in autokatalytischen Reaktionskaskaden, wo auch nur eine minimale Menge von anfänglichen Leck wird schließlich in die vollständige Aktivierung des Kaskaden 19,20 führen offensichtlichsten. Umgekehrt Reaktionen oft nicht zu dem erwarteten Ausmaß der Aktivierung zu erreichen, da einige Komponenten nicht einmal in Anwesenheit von die beabsichtigte Eingabe 7,25 auszulösen. Um die Leistung zu machen DNA basierendenNanovorrichtungen vergleichbar zu biologischen Proteinbasis benötigen solche Fehlerarten, drastisch reduziert werden. Bakterielle Plasmide oder andere biologische DNA konnte als relativ billige Quelle für hochreine DNA für Nanotechnologie-Anwendungen dienen. Große Mengen von DNA können durch Replikation in Bakterien erzeugt werden, und die Grenzkorrekturlesen Fähigkeiten lebender Systeme sorgen für die Reinheit des erhaltenen DNA. In der Tat haben einige neue Papiere den potenziellen Nutzen der biologischen DNA für Nanotechnologie-Anwendungen 21,26-28 anerkannt. Jedoch die vollständig doppelsträngige Art der Plasmid-DNA hat bisher die Verwendung als Material zur Herstellung von dynamischen DNA-Geräte, die typischerweise aus mehreren Oligonukleotiden und enthalten sowohl doppelsträngige und einzelsträngige Bereiche gestattet. In einem kürzlich erschienenen Papier 29 wurde diese Frage angesprochen und eine neue DNA-Gate-Architektur, die in erster Linie aus geklaut doppelsträngige DNA (ndsDNA) aus war einzuführend. Wichtig ist, dass Systeme der ndsDNA Tore ausgelegt sein, dass die durch jede formale chemische Reaktion Netzwerk (CRN) 29 angegeben Dynamik zu realisieren. ndsDNA Gates könnte also verwendet werden, grundsätzlich auf dynamische Systeme, die Schwingungen und Chaos, Bistabilität und Gedächtnis, Boolesche Logik oder algorithmische Verhaltensweisen 30-38 aufweisen erstellen. Zum Beispiel 29 Ref. Gezeigt, eine dreiReaktions CRN, die ein Molekular Implementierung eines &quot;Konsensus&quot; Protokoll an, das der verteilten Rechenalgorithmus 29,39,40 vorgesehen. Diese Arbeit erstmals gezeigt werden eine neue Verwendung für die CRN-Formalismus als eine &quot;Programmiersprache&quot; zur schnellen Synthese von funktionalen molekularen Systemen (Abbildung 1A). Hier ist ein detailliertes Protokoll zum Ableiten ndsDNA Gates von Plasmid-DNA ist. Zunächst ist eine Überprüfung der Sequenz Design-Prozess. Es folgt eine Erläuterung, wie synthetische Oligonukleotide enthaltendie Gate-Sequenzen in Plasmide kloniert und die Sequenz verifiziert und durch Bakterienkultur amplifiziert. Als nächstes wird gezeigt, wie ndsDNA Tore können aus den Plasmiden durch enzymatische Verarbeitung abgeleitet werden (siehe Abbildung 2). Schließlich wird ein Verfahren zum Testen von Gate Verhaltens mit Fluoreszenzkinetiken Assays beschrieben. Reaktionsmechanismus Als ein Beispiel setzt das Protokoll auf die katalytische chemische Reaktion A + B-&gt; B + C. Die Arten A, B und C (&quot;Signale&quot;, 1B) entsprechen alle einem anderen Einzelstrang-DNA-Moleküls. Die Sequenzen dieser Moleküle sind völlig unabhängig und die Stränge nicht direkt miteinander reagieren. Die Sequenzen aller Signale weisen zwei verschiedene funktionelle Domänen, das heißt, Teilsequenzen, die in Strangverdrängungsreaktionen zusammenwirken: 1) eine kurze toehold Domäne (labels ta, tb, tc), die für die Initiation der Strangverdrängung r verwendet wirdeAction, und 2) eine lange Domain (Etiketten a, b, c), die das Signal Identität bestimmt. Wechselwirkungen zwischen Signalstränge durch geklaut doppelsträngige DNA (ndsDNA) Gate-Komplexe vermittelte (genannt anmelden AB und Gabel BC) und Hilfseinzelstrang-Arten (&lt;tr r&gt;, &lt;b tr&gt;, &lt;c tb&gt; und &lt;i tc &gt;). Die formale Reaktions A + B-&gt; B + C wird durch eine Reihe von Strangverdrängungsreaktionsschritten, wobei jeder Reaktionsschritt setzt einen Ansatzpunkt für eine anschließende Reaktion (1B) ausgeführt. In diesem Beispiel werden Signale A und B sind anfänglich frei in Lösung, während das Signal C dem Gabel Gate gebunden. Am Ende der Reaktion B und C sind in Lösung. Allgemeiner Signale, die an ein Gate gebunden sind inaktiv, während Signale, die frei in Lösung aktiv sind, das heißt, sie können in einer Strangverdrängungsreaktion als teilnehmenein Eingang. Der zeitliche Verlauf der Reaktion wird unter Verwendung eines fluoreszierenden Reporterstrategie (Abbildung 1C), gefolgt. In früheren Arbeiten 29 wurde gezeigt, dass diese Reaktion Mechanismus realisiert sowohl die korrekte Stöchiometrie als auch die Kinetik der Zielreaktion.

<table><tbody><tr><td>Phusion High-Fidelity PCR Master Mix with HF Buffer</td><td>NEB</td><td>M0531S</td><td></td></tr><tr><td>PvuII-HF</td><td>NEB</td><td>R3151L</td><td></td></tr><tr><td>PstI-HF</td><td>NEB</td><td>R3140S</td><td></td></tr><tr><td>Gibson Assembly Master Mix</td><td>NEB</td><td>E2611S</td><td></td></tr><tr><td>Terrific Broth, Modified</td><td>SIGMA-ALDRICH</td><td>T0918-250G</td><td></td></tr><tr><td>QIAprep Spin Miniprep Kit (250)</td><td>QIAGEN</td><td>27106</td><td></td></tr><tr><td>QIAGEN Hispeed Maxi-prep Kit</td><td>QIAGEN</td><td>12662</td><td></td></tr><tr><td>Nb.BsrDI</td><td>NEB</td><td>R0648L</td><td></td></tr><tr><td>Nt.BstNBI</td><td>NEB</td><td>R0607L</td><td></td></tr><tr><td>NanoDrop 2000c</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Double-stranded Genomic Blocks</td><td>IDT</td><td></td><td></td></tr><tr><td>Horiba Jobin-Yvon Spex Fluorolog-3 Fluorimeter</td><td>Horiba/Jobin Yvon</td><td></td><td></td></tr><tr><td>Synthetic Quartz Cells&amp;nbsp;</td><td>Starna</td><td>23-5.45-S0G-5</td><td></td></tr><tr><td>QIAGEN Gel Extraction Kit</td><td>QIAGEN</td><td>28706</td><td></td></tr><tr><td>Plasmid Backbones</td><td>BioBrick</td><td>E0240-pSB1A2</td><td>High copy number plasmid with Ampicillin resistance. Sequence can be found from http://parts.igem.org</td></tr></tbody></table>

plasmid-derived dna strand displacement gates for implementing chemical reaction networks

DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high fidelity, is readily amplified through bacterial culture and can be stored indefinitely in the form of bacterial glycerol stocks. However, the double-stranded nature of plasmid DNA has so far hindered its efficient use for construction of DNA nanostructures or devices that typically contain single-stranded or branched domains. In recent work, it was found that nicked double stranded DNA (ndsDNA) strand displacement gates could be sourced from plasmid DNA. The following is a protocol that details how these ndsDNA gates can be efficiently encoded in plasmids and can be derived from the plasmids through a small number of enzymatic processing steps. Also given is a protocol for testing ndsDNA gates using fluorescence kinetics measurements. NdsDNA gates can be used to implement arbitrary chemical reaction networks (CRNs) and thus provide a pathway towards the use of the CRN formalism as a prescriptive molecular programming language. To demonstrate this technology, a multi-step reaction cascade with catalytic kinetics is constructed. Further it is shown that plasmid-derived components perform better than identical components assembled from synthetic DNA.

Watch this Scientific Journal Video about Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks at JoVE.com

Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks

This protocol describes a method for deriving DNA strand displacement gates from plasmids and testing them using fluorescence kinetics measurements. Gates can be modularly composed into multi-component systems to approximate the behavior of formal chemical reaction networks (CRN), demonstrating a new use for CRNs as a molecular programming language.

Plasmid stammende DNA-Strand Displacement Gatter zum Implementieren Chemical Reaction Networks

DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high ...

plasmid-derived-dna-strand-displacement-gates-for-implementing

Research

JoVE Journal

Biology

14.3K Aufrufe.  University of Washington. This protocol describes a method for deriving DNA strand displacement gates from plasmids and testing them using fluorescence kinetics measurements. Gates can be modularly composed into multi-component systems to approximate the behavior of formal chemical reaction networks (CRN), demonstrating a new use for CRNs as a molecular programming language. 

Serie: Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks

Designing a Bio-responsive Robot from DNA Origami

Nucleic acids are astonishingly versatile. In addition to their natural role as storage medium for biological information1, they can be utilized in parallel computing2,3 , recognize and bind molecular or cellular targets4,5 , catalyze chemical reactions6,7 , and generate calculated responses in a biological system8,9. Importantly, nucleic acids can be programmed to self-assemble into 2D and 3D structures10-12, enabling the integration of all these remarkable features in a single robot linking the sensing of biological cues to a preset response in order to exert a desired effect.
Creating shapes from nucleic acids was first proposed by Seeman13, and several variations on this theme have since been realized using various techniques11,12,14,15 . However, the most significant is perhaps the one proposed by Rothemund, termed scaffolded DNA origami16. In this technique, the folding of a long (&gt;7,000 bases) single-stranded DNA 'scaffold' is directed to a desired shape by hundreds of short complementary strands termed 'staples'. Folding is carried out by temperature annealing ramp. This technique was successfully demonstrated in the creation of a diverse array of 2D shapes with remarkable precision and robustness. DNA origami was later extended to 3D as well17,18 .
 
The current paper will focus on the caDNAno 2.0 software19 developed by Douglas and colleagues. caDNAno is a robust, user-friendly CAD tool enabling the design of 2D and 3D DNA origami shapes with versatile features. The design process relies on a systematic and accurate abstraction scheme for DNA structures, making it relatively straightforward and efficient.
In this paper we demonstrate the design of a DNA origami nanorobot that has been recently described20. This robot is 'robotic' in the sense that it links sensing to actuation, in order to perform a task. We explain how various sensing schemes can be integrated into the structure, and how this can be relayed to a desired effect. Finally we use Cando21 to simulate the mechanical properties of the designed shape. The concept we discuss can be adapted to multiple tasks and settings.

DNA origami is a powerful method for fabricating precise nanoscale objects by programming the self-assembly of DNA molecules. Here, we describe how DNA origami can be utilized to design a robotic robot capable of sensing biological cues and responding by shape shifting, subsequently relayed to a desired effect.

Entwerfen einer Bio-responsive Robot von DNA Origami

Nucleic acids are astonishingly versatile. In addition to their natural role as storage medium for biological information1, they can be utilized in ...

Forschung

Biotechnik

Homemade Site Directed Mutagenesis of Whole Plasmids

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned target gene can be altered by substitution, deletion or insertion of a few bases directly into a plasmid. It works by simply amplifying the whole plasmid, in a non PCR-based thermocycling reaction. During the reaction mutagenic primers, carrying the desired mutation, are integrated into the newly synthesized plasmid. In this video tutorial we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid. The protocol works with standard reagents and is independent from commercial kits, which often are very expensive. Applying this protocol can reduce the total cost of a reaction to an eighth of what it costs using some of the commercial kits. In this video we also comment on critical steps during the process and give detailed instructions on how to design the mutagenic primers.

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. Here we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid using standard reagents.

Hausgemachte Mutagenese von Whole Plasmide

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned ...

Biologie

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

Automatisierte Roboter Liquid Handling Versammlung Modular DNA-Geräte

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Using Synthetic Biology to Engineer Living Cells That Interface with Programmable Materials

We have developed an abiotic-biotic interface that allows engineered cells to control the material properties of a functionalized surface. This system is made by creating two modules: a synthetically engineered strain of E. coli cells and a functionalized material interface. Within this paper, we detail a protocol for genetically engineering selected behaviors within a strain of E. coli using molecular cloning strategies. Once developed, this strain produces elevated levels of biotin when exposed to a chemical inducer. Additionally, we detail protocols for creating two different functionalized surfaces, each of which is able to respond to cell-synthesized biotin. Taken together, we present a methodology for creating a linked, abiotic-biotic system that allows engineered cells to control material composition and assembly on nonliving substrates.

This paper presents a series of protocols for developing engineered cells and functionalized surfaces that enable synthetically engineered E. coli to control and manipulate programmable material surfaces.

Unter Verwendung der synthetischen Biologie lebenden Zellen zu entwickeln, die Schnittstelle mit programmierbarer Materialien

We have developed an abiotic-biotic interface that allows engineered cells to control the material properties of a functionalized surface. This system is ...

Design and Synthesis of a Reconfigurable DNA Accordion Rack

DNA nanostructure-based mechanical systems or DNA nanomachines, which produce complex nanoscale motion in 2D and 3D in the nanometer to &#229;ngstr&#246;m resolution, show great potential in various fields of nanotechnology such as the molecular reactors, drug delivery, and nanoplasmonic systems. The reconfigurable DNA accordion rack, which can collectively manipulate a 2D or 3D nanoscale network of elements, in multiple stages in response to the DNA inputs, is described. The platform has potential to increase the number of elements that DNA nanomachines can control from a few elements to a network scale with multiple stages of reconfiguration.
In this protocol, we describe the entire experimental process of the reconfigurable DNA accordion rack of 6 by 6 meshes. The protocol includes a design rule and simulation procedure of the structures and a wet-lab experiment for synthesis and reconfiguration. In addition, analysis of the structure using TEM (transmission electron microscopy) and FRET (fluorescence resonance energy transfer) is included in the protocol. The novel design and simulation methods covered in this protocol will assist researchers to use the DNA accordion rack for further applications.

We describe the detailed protocol for design, simulation, wet-lab experiments, and analysis for a reconfigurable DNA accordion rack of 6 by 6 meshes.

Design und Synthese eines rekonfigurierbaren DNA-Akkordeon-Racks

DNA nanostructure-based mechanical systems or DNA nanomachines, which produce complex nanoscale motion in 2D and 3D in the nanometer to &#229;ngstr&#246;m ...

Functional Surface-immobilization of Genes Using Multistep Strand Displacement Lithography

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic bioreactor system. In contrast to other approaches towards artificial cellular systems, such a setup allows for a continuous supply with gene expression reagents and simultaneous draining of waste products. This facilitates the implementation of cell-free gene expression processes over extended periods of time, which is important for the realization of dynamic gene regulatory feedback systems. Here we provide a detailed protocol for the fabrication of genetic biochips using a simple-to-use lithographic technique based on DNA strand displacement reactions, which exclusively uses commercially available components. We also provide a protocol on the integration of compartmentalized genes with a polydimethylsiloxane (PDMS)-based microfluidic system. Furthermore, we show that the system is compatible with total internal reflection fluorescence (TIRF) microscopy, which can be used for the direct observation of molecular interactions between DNA and molecules contained in the expression mix.

We describe a simple lithographic procedure for the immobilization of gene-length DNA molecules on a surface, which can be used to perform cell-free gene expression experiments on biochips.

Funktionale Oberfläche-Immobilisierung von Genen mit mehrstufigen Strang Verschiebung Lithographie

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic ...

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that cut outside of their recognition sites and create a short overhang. This modular cloning (MoClo) system uses a hierarchical workflow in which different DNA parts, such as promoters, coding sequences (CDS), and terminators, are first cloned into an entry vector. Multiple entry vectors then assemble into transcription units. Several transcription units then connect into a multi-gene plasmid. The Golden Gate cloning strategy is of tremendous advantage because it allows scar-less, directional, and modular assembly in a one-pot reaction. The hierarchical workflow typically enables the facile cloning of a large variety of multi-gene constructs with no need for sequencing beyond entry vectors. The use of fluorescent protein dropouts enables easy visual screening. This work provides a detailed, step-by-step protocol for assembling multi-gene plasmids using the yeast modular cloning (MoClo) kit. We show optimal and suboptimal results of multi-gene plasmid assembly and provide a guide for screening for colonies. This cloning strategy is highly applicable for yeast metabolic engineering and other situations in which multi-gene plasmid cloning is required.

The goal of this protocol is to provide a detailed, step-by-step guide for assembling multi-gene constructs using the modular cloning system based on Golden Gate cloning. It also gives recommendations on critical steps to ensure optimal assembly based on our experiences.

Schnelle Montage von Multi-Gen-Konstrukten mit modularem Golden Gate Klonen

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that ...

DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation

DNA nanotechnology enables programmable self-assembly of nucleic acids into user-prescribed shapes and dynamics for diverse applications. This work demonstrates that concepts from DNA nanotechnology can be used to program the enzymatic activity of the phage-derived T7 RNA polymerase (RNAP) and build scalable synthetic gene regulatory networks. First, an oligonucleotide-tethered T7 RNAP is engineered via expression of an N-terminally SNAP-tagged RNAP and subsequent chemical coupling of the SNAP-tag with a benzylguanine (BG)-modified oligonucleotide. Next, nucleic-acid strand displacement is used to program polymerase transcription on-demand. In addition, auxiliary nucleic acid assemblies can be used as &#34;artificial transcription factors&#34; to regulate the interactions between the DNA-programmed T7 RNAP with its DNA templates. This in vitro transcription regulatory mechanism can implement a variety of circuit behaviors such as digital logic, feedback, cascading, and multiplexing. The composability of this gene regulatory architecture facilitates design abstraction, standardization, and scaling. These features will enable the rapid prototyping of in vitro&#160;genetic devices for applications such as bio-sensing, disease detection, and data storage.

We describe the engineering of a novel DNA-tethered T7 RNA polymerase to regulate in vitro transcription reactions. We discuss the steps for protein synthesis and characterization, validate proof-of-concept transcriptional regulation, and discuss its applications in molecular computing, diagnostics, and molecular information processing.

DNA-Tethered RNA Polymerase für programmierbare In-vitro-Transkription und molekulare Berechnung

DNA nanotechnology enables programmable self-assembly of nucleic acids into user-prescribed shapes and dynamics for diverse applications. This work ...

Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins

Synthetic gene Boolean gates and digital circuits have a broad range of applications, from medical diagnostics to environmental care. The discovery of the CRISPR-Cas systems and their natural inhibitors-the anti-CRISPR proteins (Acrs)-provides a new tool to design and implement in vivo gene digital circuits. Here, we describe a protocol that follows the idea of the "Design-Build-Test-Learn" biological engineering cycle and makes use of dCas9/dCas12a together with their corresponding Acrs to establish small transcriptional networks, some of which behave like Boolean gates, in Saccharomyces cerevisiae. These results point out the properties of dCas9/dCas12a as transcription factors. In particular, to achieve maximal activation of gene expression, dSpCas9 needs to interact with an engineered scaffold RNA that collects multiple copies of the VP64 activation domain (AD). In contrast, dCas12a shall be fused, at the C terminus, with the strong VP64-p65-Rta (VPR) AD. Furthermore, the activity of both Cas proteins is not enhanced by increasing the amount of sgRNA/crRNA in the cell. This article also explains how to build Boolean gates based on the CRISPR-dCas-Acr interaction. The AcrIIA4 fused hormone-binding domain of the human estrogen receptor is the core of a NOT gate responsive to &#946;-estradiol, whereas AcrVAs synthesized by the inducible GAL1 promoter permits to mimic both YES and NOT gates with galactose as an input. In the latter circuits, AcrVA5, together with dLbCas12a, showed the best logic behavior.

CRISPR-Cas systems and anti-CRISPR proteins were integrated into the scheme of Boolean gates in Saccharomyces cerevisiae. The new small logic circuits showed good performance and deepened the understanding of both dCas9/dCas12a-based transcription factors and the properties of anti-CRISPR proteins.

Digitale Genschaltkreise auf Basis von CRISPR-Cas-Systemen und Anti-CRISPR-Proteinen

Synthetic gene Boolean gates and digital circuits have a broad range of applications, from medical diagnostics to environmental care. The discovery of the ...

Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1&#945; Promoter Using Gibson Assembly

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized plasmids for expression of exogenous genes in mouse embryonic stem cells (mESCs). Expression of exogenous genes under the control of the SV40 or human cytomegalovirus promoters diminishes quickly after transfection into mESCs. A remedy for this diminished expression is to use the human elongation factor-1 alpha (hEF1&#945;) promoter to drive gene expression. Plasmid vectors containing hEF1&#945; are not as widely available as SV40- or CMV-containing plasmids, especially those also containing N-terminal 3xFLAG-tags. The protocol described here is a rapid method to create plasmids expressing FLAG-tagged CstF-64 and CstF-64 mutant under the expressional regulation of the hEF1&#945; promoter. GA uses a blend of DNA exonuclease, DNA polymerase and DNA ligase to make cloning of overlapping ends of DNA fragments possible. Based on the template DNAs we had available, we designed our constructs to be assembled into a single sequence. Our design used four DNA fragments: pcDNA 3.1 vector backbone, hEF1&#945; promoter part 1, hEF1&#945; promoter part 2 (which contained 3xFLAG-tag purchased as a double-stranded synthetic DNA fragment), and either CstF-64 or specific CstF-64 mutant. The sequences of these fragments were uploaded to a primer generation tool to design appropriate PCR primers for generating the DNA fragments. After PCR, DNA fragments were mixed with the vector containing the selective marker and the GA cloning reaction was assembled. Plasmids from individual transformed bacterial colonies were isolated. Initial screen of the plasmids was done by restriction digestion, followed by sequencing. In conclusion, GA allowed us to create customized plasmids for gene expression in 5 days, including construct screens and verification.

Synthesis of custom plasmids is labor and time consuming. This protocol describes the use of Gibson assembly cloning to reduce the work and duration of custom DNA cloning procedure. The protocol described also produces reliable tagged protein constructs for mammalian expression at similar cost to the traditional cut-and-paste DNA cloning.

Erzeugung von Plasmid-Vektoren, die FLAG-markierten Proteine ​​Nach der Verordnung der Menschen Elongation Factor-1α Promoter Mit Gibson Assembly

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized ...

Plasmid stammende DNA-Strand Displacement Gatter zum Implementieren Chemical Reaction Networks

Zusammenfassung

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Automatisierte Roboter Liquid Handling Versammlung Modular DNA-Geräte

Unter Verwendung der synthetischen Biologie lebenden Zellen zu entwickeln, die Schnittstelle mit programmierbarer Materialien

Design und Synthese eines rekonfigurierbaren DNA-Akkordeon-Racks

Funktionale Oberfläche-Immobilisierung von Genen mit mehrstufigen Strang Verschiebung Lithographie

Schnelle Montage von Multi-Gen-Konstrukten mit modularem Golden Gate Klonen

DNA-Tethered RNA Polymerase für programmierbare In-vitro-Transkription und molekulare Berechnung

Digitale Genschaltkreise auf Basis von CRISPR-Cas-Systemen und Anti-CRISPR-Proteinen

Erzeugung von Plasmid-Vektoren, die FLAG-markierten Proteine Nach der Verordnung der Menschen Elongation Factor-1α Promoter Mit Gibson Assembly

Plasmid stammende DNA-Strand Displacement Gatter zum Implementieren Chemical Reaction Networks

Zusammenfassung

Weitere Videos entdecken

Kapitel in diesem Video

Entwerfen einer Bio-responsive Robot von DNA Origami

Hausgemachte Mutagenese von Whole Plasmide

Automatisierte Roboter Liquid Handling Versammlung Modular DNA-Geräte

Unter Verwendung der synthetischen Biologie lebenden Zellen zu entwickeln, die Schnittstelle mit programmierbarer Materialien

Design und Synthese eines rekonfigurierbaren DNA-Akkordeon-Racks

Funktionale Oberfläche-Immobilisierung von Genen mit mehrstufigen Strang Verschiebung Lithographie

Schnelle Montage von Multi-Gen-Konstrukten mit modularem Golden Gate Klonen

DNA-Tethered RNA Polymerase für programmierbare In-vitro-Transkription und molekulare Berechnung

Digitale Genschaltkreise auf Basis von CRISPR-Cas-Systemen und Anti-CRISPR-Proteinen

Erzeugung von Plasmid-Vektoren, die FLAG-markierten Proteine ​​Nach der Verordnung der Menschen Elongation Factor-1α Promoter Mit Gibson Assembly

Erzeugung von Plasmid-Vektoren, die FLAG-markierten Proteine Nach der Verordnung der Menschen Elongation Factor-1α Promoter Mit Gibson Assembly