Figuras 1, 2, 3, 4, 6, 8 y Tablas 2, 3, 10, 15, 16 se modifican de la referencia 29. Este trabajo fue apoyado por la Fundación Nacional de Ciencia (NSF conceder-CCF 1.117.143 y NSF-CCF 1.162.141 de GS). Y.-JC fue apoyado por becas del Gobierno de Taiwán. SDR fue apoyada por el Programa Nacional Science Foundation Graduate Research Fellowship (GRFP).

<ol>
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The authors declare no competing financial interests.

Este documento describe un método para derivar puertas ndsDNA a partir de ADN plásmido de alta pureza. Por otra parte, un protocolo se presenta para caracterizar el rendimiento de puerta usando un ensayo de la cinética de fluorescencia. Los datos experimentales muestran que el sistema de plásmido derivado supera a su contraparte sintética incluso si el sistema sintético se ensambla a partir de hebras purificó usando electroforesis en gel de poliacrilamida (PAGE). Probablemente, el mejor desempeño de puertas plásmido derivado se debe principalmente a la muy alta pureza del ADN biológica. ADN sintético contiene una variedad de errores, en supresiones particulares que resultan en oligonucleótidos de longitud n-1, y dichos productos secundarios son normalmente no se elimina por completo en PAGE o cromatografía líquida de alta resolución (HPLC) procedimientos de purificación. También se observaron mejoras similares a los reportados aquí en un estudio previo de un amplificador de horquilla catalizada que utiliza ADN derivado de fuentes biológicas 21. <p clculo = &quot;jove_content&quot;&gt; Sin embargo, incluso el uso de puertas de plásmido derivado no puede eliminar por completo los errores en el desempeño de puerta, para los que hay al menos dos razones: en primer lugar el exceso de la digestión o la falta de corte de precisión puede conducir a puertas con demasiados muescas o hendiduras en las posiciones equivocadas. En cualquier caso, las puertas son más propensos a participar en reacciones no deseadas. Tales problemas pueden aliviarse mediante la optimización de la cantidad de enzima utilizada (véase la Figura 4). En segundo lugar, en estos experimentos, la mayoría de las entradas y las hebras eran auxiliares ADN sintético y de este modo contenían deleciones y mutaciones. En principio, todas las entradas de una sola hebra y hebras auxiliares también se pueden obtener a partir de ADN fagémido a través de una digestión con enzimas de nicking del genoma viral m13 pre-codificada 26. Tal vez el funcionamiento del circuito se puede mejorar adicionalmente usando ssDNA derivado del genoma bacteriano. Mientras que se encontró el uso de puertas de plásmidos derivados para mejorar el rendimiento del circuito, un análisisde los tiempos de procesamiento de coste y reveló que mientras la producción de puertas de plásmido derivado es ligeramente más barato (Tabla 15), se tarda 2-3 veces más largo el tiempo de procesamiento en comparación con el montaje y la purificación de puertas a partir de oligos sintetizados comercialmente (Tabla 16). Los costes primarios de puertas plásmido derivado son la síntesis de genes y el uso de enzimas de restricción. Por 300 pmoles de puertas (suficientes para 15 reacciones en 30 nM), el costo estimado para Únete puertas es de aproximadamente $ 170 y $ 200 para Fork puertas, la diferencia de costos se debe a la utilización de diferentes enzimas mellar. Por el contrario, la síntesis química de los hilos para los mismos costes de compuerta alrededor de $ 260 incluyendo una cuota de purificación PÁGINA. El costo de tiempo principal para puertas plásmido derivado está en el procedimiento de clonación, que, al igual que la síntesis de ADN, puede ser subcontratado a una empresa de síntesis de genes. Sin embargo, una vez ensamblados, puertas plásmido derivado tienen la ventaja de que los plásmidos de acogida fácilmente se pueden replicar unand se puede almacenar en forma de stocks de glicerol bacterianas. Esto hace posible la reutilización de las puertas muchas veces. De cara al futuro, la mejora del rendimiento de las puertas de plásmidos derivados podría permitir a una gama mucho más amplia de la dinámica de los comportamientos que han sido experimentalmente demostrado hasta ahora con ADN CRN. Por ejemplo, la reciente 47,48 trabajo teórico sugiere que los patrones espaciales auto-organizados en la escala macro se pueden realizar con el ADN CRNs a través de un mecanismo de difusión de reacción. El método que aquí se presenta proporciona una ruta viable para la construcción de los componentes moleculares subyacentes para tales materiales de ADN auto-patrones. Aunque difícil, el desarrollo de morfologías escala macro de una manera programable tendría implicaciones significativas en áreas que van desde la investigación de biomateriales para la medicina regenerativa.

La previsibilidad de Watson-Crick ha permitido a la nanotecnología de ADN dinámico para emerger como una forma programable para diseñar dispositivos moleculares con propiedades dinámicas 1,2. En particular, cadena de ADN desplazamiento - una reacción de hibridación competitiva programable - ha demostrado ser un poderoso mecanismo para la ingeniería de sistemas dinámicos de ADN. En una reacción de desplazamiento de cadena de ADN, un oligonucleótido entrante desplaza una &quot;salida&quot; hebra previamente atado de una pareja de unión complementaria. Múltiples tales reacciones pueden ser encadenados juntos en cascadas de reacción de varios pasos con un alto grado de control sobre el orden y el tiempo de reacción individual 3 pasos. ADN cascadas de desplazamiento de cadena se han utilizado para crear circuitos digitales y analógicas moleculares 4-7, 8-10 nanoestructuras conmutables, motores moleculares autónomas 11-15, y amplificadores catalíticos no covalentes 13,16-21. Por otra parte, DDispositivos NA utilizando reacciones de desplazamiento de hebra se pueden simular y diseñados para diversas aplicaciones que usan el ordenador con ayuda de software de diseño 22-24. Actualmente, el ADN sintetizado químicamente sirve como material principal para la nanotecnología de ADN. Sin embargo, los errores en el proceso de la síntesis de ADN, y los oligonucleótidos resultantes imperfectos, se cree que limitar el rendimiento de los dispositivos dinámicos de ADN al causar reacciones secundarias erróneos. Por ejemplo, las reacciones de &quot;fugas&quot; pueden resultar en la liberación de un oligonucleótido de salida incluso en la ausencia de un disparador de reacción. Estos efectos son más evidentes en las cascadas de reacción autocatalíticos donde incluso una cantidad mínima de fuga inicial con el tiempo como resultado la activación completa del 19,20 cascada. Por el contrario, las reacciones a menudo fallan en alcanzar el nivel esperado de activación debido a que algunos componentes no activan incluso en la presencia de la entrada prevista 7,25. Para hacer que el rendimiento de ADN a base denanodispositivos comparables a sus homólogos basados ​​en proteínas biológicas, tales modos de error deben reducirse dramáticamente. Plásmidos bacterianos u otro ADN biológica podrían servir como una fuente relativamente barata de ADN de alta pureza para aplicaciones de la nanotecnología. Grandes cantidades de ADN se pueden generar por la replicación en bacterias y las capacidades de corrección de pruebas intrínsecas de los sistemas vivos asegurar la pureza del ADN resultante. De hecho, varios estudios recientes han reconocido la utilidad potencial de ADN biológico para aplicaciones de la nanotecnología 21,26-28. Sin embargo, la naturaleza completamente de doble hebra de ADN del plásmido hasta ahora ha prohibido su uso como material para la fabricación de dispositivos dinámicos de ADN, que normalmente consisten en múltiples oligonucleótidos y contienen ambos dominios de doble cadena y de cadena sencilla. En un trabajo reciente 29 de este tema fue abordado y una nueva arquitectura de puerta de ADN que se compone principalmente de ADN de doble cadena mellado (ndsDNA) fue introducirre. Es importante destacar que los sistemas de puertas ndsDNA pueden ser diseñados que dan cuenta de la dinámica especificados por cualquier red reacción química formales (CRN) 29. puertas ndsDNA tanto, se podrían utilizar, en principio, para crear sistemas dinámicos que presentan oscilaciones y caos, biestabilidad y la memoria, la lógica de Boole o comportamientos algorítmicos 30-38. Por ejemplo, Ref. 29 demostraron una CRN de tres reacción que proporciona una implementación molecular de un protocolo de &quot;consenso&quot;, un tipo de algoritmo de computación distribuida 29,39,40. Este trabajo demuestra por primera vez un nuevo uso para el formalismo CRN como un &quot;lenguaje de programación&quot; para sintetizar rápidamente los sistemas moleculares funcionales (Figura 1). Aquí, se proporciona un protocolo detallado para derivar puertas ndsDNA de ADN plásmido. Primero es una revisión del proceso de diseño de la secuencia. Luego sigue una explicación de cómo oligonucleótidos sintéticos que contienenlas secuencias de compuerta se clonaron en plásmidos y la secuencia verificados y amplificados a través de cultivo bacteriano. A continuación, se muestra cómo ndsDNA puertas se pueden derivar de los plásmidos mediante procesamiento enzimático (véase la Figura 2). Finalmente, un método para probar el comportamiento puerta utilizando ensayos de cinética de fluorescencia se describe. Mecanismo de reacción Como ejemplo, el protocolo se centra en el catalítica reacción química A + B-&gt; B + C. La especie A, B, y C (&quot;señales&quot;, Figura 1B) todos corresponden a una molécula de ADN de una sola hebra diferente. Las secuencias de estas moléculas son completamente independientes y las hebras no reaccionan entre sí directamente. Las secuencias de todas las señales tienen dos dominios funcionales diferentes, es decir, subsecuencias que actúan juntos en reacciones de desplazamiento de hebra: 1) un dominio de punto de apoyo corto (etiquetas ta, tb, tc) que se utiliza para la iniciación de desplazamiento de cadena reaction, y 2) un dominio de largo (etiquetas a, b, c) que determina la identidad de la señal. Las interacciones entre los hilos de señal están mediadas por ADN (ndsDNA) complejos de puerta de doble cadena con muescas (llamados Únete AB y Tenedor aC) y las especies de cadena sencilla auxiliares (&lt;tr r&gt;, &lt;b tr&gt;, &lt;c tb&gt; y &lt;i tc &gt;). La reacción A + B- formales&gt; B + C se ejecuta a través de una serie de etapas de reacción de desplazamiento de cadena, donde cada paso de reacción expone un punto de apoyo para una reacción posterior (Figura 1B). En este ejemplo, las señales A y B son inicialmente libre en solución, mientras que la señal C está unido a la puerta de tenedor. Al final de la reacción B y C están en solución. De manera más general, las señales que están obligados a una puerta están inactivas mientras que las señales que están libres en solución son activos, es decir, que pueden participar en una reacción de desplazamiento de cadena comouna entrada. El curso temporal de la reacción se sigue utilizando una estrategia de indicador fluorescente (Figura 1C). En un trabajo anterior 29, se demostró que este mecanismo de reacción no sólo da cuenta de la estequiometría correcta, sino también la cinética de la reacción objetivo.

<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.

Plásmido derivado de ADN Strand Displacement Puertas de Redes de reacción de Ejecución Química

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 vistas.  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.

Diseño de un Robot Bio-respuesta a partir del ADN Origami

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

Investigación

Bioingeniería

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.

Mutagénesis dirigida casera de plásmidos enteros

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

Biología

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.

Automatizado de montaje robotizada manipulación líquido de dispositivos modulares de la 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.

El uso de la biología sintética para Ingeniero células vivas que interactúan con materiales programables

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.

Diseño y síntesis de un ADN Reconfigurable acordeón 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 ...

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-Inmovilización funcional de Genes utilizando litografía de desplazamiento de varias fases de la cadena

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.

Montaje rápido de construcciones multigénicas mediante clonación modular 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.

ARN polimerasa atado al ADN para transcripción in vitro programable y computación molecular

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.

Circuitos digitales genéticos basados en sistemas CRISPR-Cas y proteínas 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 ...

Mouse Genome Engineering Using Designer Nucleases

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well as for modeling human diseases and testing therapeutic strategies. Recent advances in the use of designer nucleases such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system for site-specific genome engineering open the possibility to perform rapid targeted genome modification in virtually any laboratory species without the need to rely on embryonic stem (ES) cell technology. A genome editing experiment typically starts with identification of designer nuclease target sites within a gene of interest followed by construction of custom DNA-binding domains to direct nuclease activity to the investigator-defined genomic locus. Designer nuclease plasmids are in vitro transcribed to generate mRNA for microinjection of fertilized mouse oocytes. Here, we provide a protocol for achieving targeted genome modification by direct injection of TALEN mRNA into fertilized mouse oocytes.

Designer nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) can be used to modify the genome of mouse preimplantation embryos by triggering both the nonhomologous end joining (NHEJ) and homologous recombination (HR) pathways. These advances enable the rapid generation of mice with precise genetic modifications.

Del genoma del ratón Ingeniería Utilizando Nucleasas Designer

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well ...