Figure 1, 2, 3, 4, 6, 8 e tabelle 2, 3, 10, 15, 16 vengono modificati dal Ref 29. Questo lavoro è stato sostenuto dal National Science Foundation (NSF concedere-CCF 1.117.143 e NSF-CCF 1.162.141 a GS). Y.-JC è stata sostenuta da borse di studio del governo di Taiwan. SDR è stato sostenuto dalla Science Foundation Graduate Research Fellowship Program Nazionale (GRFP).

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The authors declare no competing financial interests.

Questo documento descrive un metodo per derivare cancelli ndsDNA da DNA plasmidico altamente puro. Inoltre, un protocollo è presentato per caratterizzare prestazioni cancello usando un test cinetica di fluorescenza. Dati sperimentali mostrano che il sistema plasmide derivato supera la sua controparte sintetica anche se il sistema di sintesi viene assemblato con trefoli purificato usando SDS-PAGE (PAGINA). Probabilmente, il miglioramento delle prestazioni di cancelli plasmide derivato è dovuta principalmente alla elevatissima purezza del DNA biologica. DNA sintetico contiene una varietà di errori, in particolare delezioni che provocano oligonucleotidi di lunghezza tali prodotti collaterali n-1, e sono tipicamente non completamente rimosso in PAGE o cromatografia liquida ad alta prestazione (HPLC) procedure di purificazione. Sono stati inoltre osservati miglioramenti simili a quelli riportati qui in un precedente studio di un amplificatore tornante catalizzata che usati DNA di origine biologica 21. <p class = &quot;jove_content&quot;&gt; Tuttavia, anche l&#39;utilizzo di porte plasmide derivato non può eliminare completamente gli errori nell&#39;esecuzione porta, per cui ci sono almeno due motivi: il primo over-digestione o la mancanza di precisione di taglio può portare a cancelli con troppe scheggiature o nick nelle posizioni sbagliate. In entrambi i casi, i gate sono più probabilità di partecipare a reazioni indesiderate. Tali problemi possono essere alleviati ottimizzando la quantità di enzima utilizzato (vedere Figura 4). In secondo luogo, in questi esperimenti, la maggior parte degli ingressi e trefoli ausiliari erano DNA sintetico e quindi contenevano delezioni e mutazioni. In linea di principio, tutti gli input a singolo filamento e fili supplementari potrebbero essere ottenuti anche dal DNA fagemide attraverso un enzima digestione intaccare del genoma virale M13 pre-codificato 26. Forse le prestazioni del circuito può essere ulteriormente migliorata utilizzando ssDNA derivata dal genoma batterico. Mentre è stato trovato l&#39;uso di porte plasmidi derivati ​​per migliorare le prestazioni del circuito, un&#39;analisidei costi e tempi di lavorazione rivelato che mentre la produzione di porte plasmide derivato è leggermente più economico (Tabella 15), richiede 2-3 volte più lungo tempo di lavorazione rispetto al montaggio e purificazione di porte da oligonucleotidi sintetizzati in commercio (Tabella 16). I costi primari di porte plasmide derivato sono sintesi genica e l&#39;impiego di enzimi di restrizione. Per 300 pmoli di cancelli (sufficienti per 15 reazioni 30 nm), il costo stimato per Iscriviti al cancello è di circa $ 170 e $ 200 per Forcella cancelli, la differenza di costo è dovuta all&#39;utilizzo di diversi enzimi intaccare. Al contrario, la sintesi chimica dei trefoli per gli stessi costi di gate intorno $ 260 compresa una commissione purificazione PAGE. Il costo orario principale per cancelli plasmide derivato è nella procedura di clonazione, che, come la sintesi del DNA, può essere affidata a una società di sintesi gene. Tuttavia, una volta assemblati, cancelli plasmide derivato hanno il vantaggio che i plasmidi ospitanti possono facilmente essere replicate unnd possono essere memorizzati in forma di scorte glicerolo batteriche. Ciò consente di riutilizzare le porte più volte. Guardando al futuro, il miglioramento delle prestazioni dei cancelli plasmide-derivato potrebbe consentire una gamma molto più ampia di dinamica dei comportamenti che sono stati sperimentalmente dimostrato finora con CRNS DNA. Ad esempio, di recente 47,48 lavoro teorico ha suggerito che i modelli spaziali autorganizzati alla macro-scala possono essere realizzate con DNA CRNS attraverso un meccanismo di diffusione di reazione. Il metodo qui presentato fornisce un percorso praticabile per costruire i componenti molecolari sottostanti per tali materiali DNA auto-patterning. Anche se impegnativo, lo sviluppo di morfologie macro-scala in modo programmabile avrebbe implicazioni significative in settori che vanno dalla ricerca biomateriali alla medicina rigenerativa.

La prevedibilità di Watson-Crick base di abbinamento è permesso nanotecnologia del DNA dinamico di emergere come un modo programmabile per la progettazione di dispositivi molecolari con proprietà dinamiche 1,2. In particolare, lo spostamento filamento di DNA - una reazione di ibridazione competitiva programmabile - ha dimostrato di essere un potente meccanismo di ingegneria dei sistemi dinamici di DNA. In una reazione spostamento filamento di DNA, un oligonucleotide arrivo sposta una ciocca precedentemente legato &quot;uscita&quot; da un partner di legame complementare. Molteplici tali reazioni possono essere concatenati in cascate di reazione multi-step con un alto grado di controllo sull&#39;ordine e la tempistica della reazione individuale passi 3. DNA cascate spostamento filone sono stati utilizzati per creare circuiti digitali e analogici molecolari 4-7, 8-10 nanostrutture commutabili, motori molecolari autonome 11-15, e non covalenti amplificatori catalitiche 13,16-21. Inoltre, DDispositivi NA utilizzando reazioni di spostamento dei filamenti possono essere simulati e progettati per diverse applicazioni utilizzando il software di progettazione assistita da computer 22-24. Attualmente, il DNA sintetizzato chimicamente serve come il materiale principale per le nanotecnologie del DNA. Tuttavia, gli errori nel processo di sintesi del DNA, e le conseguenti oligonucleotidi imperfetti, si ritiene che limitare le prestazioni dei dispositivi di Dynamic DNA, provocando reazioni collaterali errati. Ad esempio, reazioni &quot;fuga&quot; possono causare il rilascio di un oligonucleotide uscita anche in assenza di un trigger reazione. Tali effetti sono più evidenti in cascate di reazione autocatalitiche dove anche una minima quantità di perdita iniziale eventualmente sfociare nella piena attivazione della cascata 19,20. Viceversa, reazioni spesso non riescono a raggiungere il livello atteso di attivazione perché alcuni componenti non attivano anche in presenza dell&#39;ingresso previsto 7,25. Per rendere le prestazioni di DNA-basednanodispositivi paragonabili alle loro controparti a base di proteine ​​biologiche, tali modalità di errore devono essere drasticamente ridotto. Plasmidi batterici o altro DNA biologico potrebbe servire come fonte relativamente a buon mercato di DNA altamente puro per applicazioni nanotecnologiche. Grandi quantità di DNA possono essere generati dalla replica nei batteri e le capacità di correzione di bozze intrinseche dei sistemi viventi garantiscono la purezza del DNA risultante. In effetti, diversi lavori recenti hanno riconosciuto la potenziale utilità del DNA biologico per applicazioni nanotecnologiche 21,26-28. Tuttavia, la natura completamente a doppio filamento di DNA plasmide ha finora vietato l&#39;uso come materiale per la realizzazione di dispositivi di DNA dinamici, normalmente rappresentato più oligonucleotidi e contengono entrambi i domini doppio filamento e singolo filamento. In un recente lavoro 29 questo problema è stato affrontato e una nuova architettura porta DNA che consiste principalmente di intaccate DNA a doppia elica (ndsDNA) era introdurred. È importante sottolineare che i sistemi di porte ndsDNA possono essere progettati che realizzano le dinamiche specificato da una rete formale reazione chimica (CRN) 29. cancelli ndsDNA potrebbero quindi essere utilizzati, in linea di principio, di creare sistemi dinamici che presentano oscillazioni e il caos, bistabilità e la memoria, la logica booleana o comportamenti algoritmici 30-38. Ad esempio, Rif. ​​29 hanno dimostrato una CRN tre reazione che ha fornito un&#39;implementazione molecolare di un protocollo &quot;consenso&quot;, un tipo di algoritmo di calcolo distribuito 29,39,40. Questo primo lavoro ha dimostrato un nuovo uso per il formalismo CRN come un &quot;linguaggio di programmazione&quot; per la rapida sintesi di sistemi molecolari funzionali (Figura 1A). Qui, è previsto un protocollo dettagliato per ricavare porte ndsDNA dal plasmide DNA. Il primo è una revisione del processo di progettazione sequenza. Segue una spiegazione di oligonucleotidi sintetici contenenti comele sequenze del cancello vengono clonati in plasmidi e la sequenza verificate e amplificati tramite coltura batterica. Successivamente, è mostrato come ndsDNA porte possono essere derivati ​​da plasmidi di trattamento enzimatico (vedere Figura 2). Infine, un metodo per testare il comportamento cancello utilizzando saggi cinetici fluorescenza è delineato. Meccanismo di reazione Come esempio, il protocollo si concentra sulla reazione chimica catalitica A + B-&gt; B + C. Le specie A, B, e C (&quot;segnali&quot;, Figura 1B) tutti corrispondono ad una diversa molecola di DNA a singolo filamento. Le sequenze di queste molecole sono completamente indipendenti ed i fili non reagiscono uno con l&#39;altro direttamente. Le sequenze di tutti i segnali hanno due differenti domini funzionali, cioè sottosequenze che agiscono insieme a reazioni di spostamento strand: 1) un punto d&#39;appoggio a dominio breve (etichette ta, tb, tc) che viene utilizzato per l&#39;avvio di filo spostamento reaction, e 2) una lunga dominio (etichette a, b, c), che determina l&#39;identità del segnale. Le interazioni tra fili del segnale sono mediati da intaccate DNA (ndsDNA) complessi a doppia elica del cancello (chiamati Join AB e forcella aC) e ausiliari di specie a singolo filamento (&lt;tr r&gt;, &lt;b tr&gt;, &lt;c tb&gt; e &lt;i tc &gt;). La reazione A + B- formale&gt; B + C viene eseguito attraverso una serie di stadi di reazione di spostamento del filo, in cui ogni stadio di reazione espone un punto d&#39;appoggio per una successiva reazione (Figura 1B). In questo esempio, i segnali A e B sono inizialmente libere in soluzione mentre il segnale C è legato al cancello forcella. Al termine della reazione B e C sono in soluzione. Più in generale, i segnali che sono associati a un cancello sono inattive mentre i segnali che sono liberi in soluzione sono attivi, cioè, essi possono partecipare a una reazione di spostamento del filo comeun ingresso. Il decorso della reazione viene seguito mediante una strategia reporter fluorescente (Figura 1C). Nel precedente lavoro 29, è stato dimostrato che questo meccanismo di reazione realizza non solo la stechiometria corretta ma anche la cinetica della reazione di destinazione.

<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.

Plasmidi derivati ​​da DNA Strand Displacement Cancelli per reti di reazione implementazione chimici

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

Universidad San Sebastian

Research

JoVE Journal

Biology

14.3K Views.  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. 

Video: 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.

Progettazione di un robot bio-sensibile da DNA Origami

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

Ricerca

Bioingegneria

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.

Mutagenesi sito fatto in casa Regia di Plasmidi Whole

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

Biologia

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.

Automatizzato robot Liquid Handling Assembly di dispositivi modulari del DNA

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.

Utilizzando biologia sintetica per Ingegnere cellule viventi che si interfacciano con materiali programmabili

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.

Progettazione e sintesi di un Rack di fisarmonica di DNA riconfigurabile

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.

Superficie-immobilizzazione funzionale dei geni usando Multistep Strand Displacement Litografia

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.

Assemblaggio rapido di costrutti multi-gene utilizzando la clonazione modulare del Golden Gate

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.

RNA polimerasi legata al DNA per la trascrizione in vitro programmabile e il calcolo molecolare

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.

Circuiti digitali genici basati su sistemi CRISPR-Cas e proteine anti-CRISPR

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.

Generazione di plasmidi vettori che esprimono proteine ​​FLAG-tagged Ai sensi del regolamento di Human Factor-allungamento 1α Promotore Uso Gibson Assembly

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

Plasmidi derivati da DNA Strand Displacement Cancelli per reti di reazione implementazione chimici

Riepilogo

Esplora Altri Video

Progettazione di un robot bio-sensibile da DNA Origami

Mutagenesi sito fatto in casa Regia di Plasmidi Whole

Automatizzato robot Liquid Handling Assembly di dispositivi modulari del DNA

Utilizzando biologia sintetica per Ingegnere cellule viventi che si interfacciano con materiali programmabili

Progettazione e sintesi di un Rack di fisarmonica di DNA riconfigurabile

Superficie-immobilizzazione funzionale dei geni usando Multistep Strand Displacement Litografia

Assemblaggio rapido di costrutti multi-gene utilizzando la clonazione modulare del Golden Gate

RNA polimerasi legata al DNA per la trascrizione in vitro programmabile e il calcolo molecolare

Circuiti digitali genici basati su sistemi CRISPR-Cas e proteine anti-CRISPR

Generazione di plasmidi vettori che esprimono proteine FLAG-tagged Ai sensi del regolamento di Human Factor-allungamento 1α Promotore Uso Gibson Assembly

Plasmidi derivati ​​da DNA Strand Displacement Cancelli per reti di reazione implementazione chimici

Riepilogo

Esplora Altri Video

Progettazione di un robot bio-sensibile da DNA Origami

Mutagenesi sito fatto in casa Regia di Plasmidi Whole

Automatizzato robot Liquid Handling Assembly di dispositivi modulari del DNA

Utilizzando biologia sintetica per Ingegnere cellule viventi che si interfacciano con materiali programmabili

Progettazione e sintesi di un Rack di fisarmonica di DNA riconfigurabile

Superficie-immobilizzazione funzionale dei geni usando Multistep Strand Displacement Litografia

Assemblaggio rapido di costrutti multi-gene utilizzando la clonazione modulare del Golden Gate

RNA polimerasi legata al DNA per la trascrizione in vitro programmabile e il calcolo molecolare

Circuiti digitali genici basati su sistemi CRISPR-Cas e proteine anti-CRISPR

Generazione di plasmidi vettori che esprimono proteine ​​FLAG-tagged Ai sensi del regolamento di Human Factor-allungamento 1α Promotore Uso Gibson Assembly

Plasmidi derivati da DNA Strand Displacement Cancelli per reti di reazione implementazione chimici

Generazione di plasmidi vettori che esprimono proteine FLAG-tagged Ai sensi del regolamento di Human Factor-allungamento 1α Promotore Uso Gibson Assembly