As Figuras 1, 2, 3, 4, 6, 8 e quadros 2, 3, 10, 15, 16 são modificados a partir de 29 Ref. Este trabalho foi financiado pela National Science Foundation (NSF conceder-CCF 1117143 e NSF-CCF 1.162.141 para GS). Y.-JC foi apoiado por bolsas de estudo governamentais de Taiwan. SDR foi apoiada pela Fundação Nacional de Ciência Programa de Pós-Graduação Research Fellowship (GRFP).

<ol>
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The authors declare no competing financial interests.

Este artigo descreve um método para derivar portões ndsDNA a partir de DNA de plasmídeo muito puro. Além disso, um protocolo é apresentada para caracterizar o desempenho portão utilizando um ensaio de cinética de fluorescência. Os dados experimentais mostram que o sistema de plasmídeo derivado de supera sua contraparte sintética, mesmo se o sistema sintético é montado a partir de fios purificado utilizando electroforese em gel de poliacrilamida (PAGE). Provavelmente, o melhor desempenho de portas derivado do plasmídeo é principalmente devido à muito alta pureza do DNA biológico. ADN sintético contém uma variedade de erros, em particular, deleções que resultam em oligonucleótidos de tais produtos secundários comprimento n-1, e são tipicamente não completamente removido em PAGE ou cromatograf ia líquida de alta eficiência (HPLC) procedimentos de purificação. Também foram observadas melhorias semelhantes aos aqui relatados em um estudo prévio de um amplificador hairpin catalisada que usou DNA derivado de fontes biológicas 21. <p class = &quot;jove_content&quot;&gt; No entanto, mesmo o uso de portas derivado do plasmídeo não pode eliminar completamente os erros no desempenho portão, para as quais existem pelo menos duas razões: em primeiro lugar sobre-digestão ou falta de precisão de corte pode levar a portões com demasiados nicks ou cortes nas posições erradas. Em qualquer caso, as portas são mais susceptíveis de participar em reacções indesejadas. Tais problemas podem ser facilitado através da optimização da quantidade de enzima utilizada (ver Figura 4). Em segundo lugar, nestas experiências, a maioria das entradas e cordões eram auxiliares de ADN sintético e continha assim as supressões e mutações. Em princípio, todas as entradas de cadeia simples e fios auxiliares também poderia ser obtido a partir de ADN de fagemideo por meio de uma digestão com enzimas de nicking do genoma viral M13 pré-codificada 26. Talvez o desempenho do circuito pode ser melhorado utilizando ADNcs derivados do genoma bacteriano. Enquanto o uso de portões derivado de plasmídeo foi encontrada para melhorar o desempenho do circuito, uma análisedos tempos de processamento e custos revelou que, enquanto a produção de portas derivado de plasmídeo é ligeiramente mais barato (Tabela 15), leva-se 2-3 vezes mais em comparação com o tempo de processamento e a purificação da montagem de portas de oligos sintetizados comercialmente (Tabela 16). Os custos primários de portões são derivados do plasmídeo síntese de genes e a utilização de enzimas de restrição. Para 300 pmol de portas (suficiente para 15 reacções em 30 nM), o custo estimado para Junte-se portas é de aproximadamente US $ 170 e US $ 200 para Fork portões, a diferença de custo ser devido ao uso de diferentes enzimas nicking. Em contraste, a síntese química dos fios para os mesmos custos de porta em torno $ 260, incluindo uma taxa de purificação PAGE. O custo do tempo primário para portas derivado do plasmídeo é no procedimento de clonagem, que, assim como a síntese de DNA, pode ser terceirizado para uma empresa de síntese de genes. No entanto, uma vez montado, portas derivado do plasmídeo tem a vantagem de que os plasmídeos hospedeiros podem ser facilmente replicadas de umaND pode ser armazenado na forma de estoques de glicerol bacterianas. Isto faz com que seja possível voltar a utilizar os portões muitas vezes. Olhando para a frente, o melhor desempenho de portas derivado do plasmídeo poderia permitir uma gama muito maior de dinâmica de comportamentos que têm sido experimentalmente demonstrado até agora com DNA CRNs. Por exemplo, recente trabalho teórico 47,48 sugeriu que padrões espaciais auto-organizadas na macro-escala podem ser realizados com DNA CRNs através de um mecanismo de difusão reação. O método aqui apresentado fornece um caminho viável para a construção de componentes moleculares subjacentes a tais materiais de DNA auto-padronização. Apesar de desafiador, desenvolvendo morfologias macro-escala de uma forma programável teria implicações significativas em áreas que vão desde pesquisas em biomateriais para a medicina regenerativa.

A previsibilidade de bases de Watson-Crick tem permitido nanotecnologia de DNA dinâmico a emergir como uma forma programável para projetar dispositivos moleculares com propriedades dinâmicas 1,2. Em particular, o deslocamento cadeia de DNA -, uma reação de hibridização competitiva programável - provou ser um poderoso mecanismo de engenharia de sistemas dinâmicos de DNA. Em uma reação de deslocamento de cadeia de DNA, um oligonucleï¿½ido de entrada desloca um &quot;output&quot; vertente anteriormente vinculado a partir de um parceiro de ligação complementar. Várias tais reações podem ser encadeados em cascatas de reação de várias etapas com um alto grau de controle sobre a ordem eo tempo de reação individual etapas 3. DNA cascatas de deslocamento de cadeia foram usadas para criar circuitos digitais e analógicos moleculares 4-7, nanoestruturas comutáveis ​​8-10, motores moleculares autónomas 11-15, e amplificadores catalíticos não covalentes 13,16-21. Além disso, DNA dispositivos que utilizam reações de deslocamento de cadeia pode ser simulado e projetado para diversas aplicações utilizando software 22-24 desenho assistido por computador. Atualmente, o DNA sintetizado quimicamente serve como o principal material para a nanotecnologia de DNA. No entanto, os erros no processo de síntese de DNA, e os oligonucleótidos imperfeitos resultantes, são acreditados para limitar o desempenho de dispositivos de ADN dinâmicas, fazendo com que as reacções laterais erradas. Por exemplo, as reacções de &quot;vazamento&quot; pode resultar na libertação de um oligonucleótido de saída, mesmo na ausência de uma reacção de disparo. Tais efeitos são mais óbvias nas cascatas de reacção autocatalítica onde mesmo uma quantidade mínima de vazamento inicial irá eventualmente resultar na activação completa da cascata 19,20. Por outro lado, as reacções muitas vezes não conseguem alcançar o nível esperado de activação porque alguns componentes não provocam, mesmo na presença de 7,25 a entrada pretendida. Para fazer com que o desempenho de DNA com base emnanodispositivos comparáveis ​​com os seus homólogos à base de proteínas biológicas, tais modos de erro precisa ser reduzido drasticamente. Plasmídeos bacterianos ou outro ADN biológica pode servir como uma fonte de ADN altamente puro relativamente barato para aplicações de nanotecnologia. Grandes quantidades de ADN pode ser gerado por replicação em bactérias e as capacidades inerentes de revisão dos sistemas vivos garantir a pureza do ADN resultante. De fato, vários trabalhos recentes têm reconhecido a utilidade potencial de DNA biológico para aplicações de nanotecnologia 21,26-28. No entanto, a natureza totalmente de cadeia dupla de DNA de plasmídeo foi medida proibida a sua utilização como um material para a fabricação de dispositivos de ADN dinâmicas, que consistem tipicamente de vários oligonucleótidos e contêm ambos os domínios de cadeia dupla e de cadeia simples. Em um recente artigo 29 esta questão foi abordada e uma nova arquitetura portão DNA que consiste principalmente de DNA de cadeia dupla entalhado (ndsDNA) foi introduzird. É importante ressaltar que os sistemas de portões ndsDNA pode ser concebido que perceber a dinâmica especificados por qualquer rede de reação química formais (CRN) 29. portões ndsDNA poderia, assim, ser utilizados, em princípio, para criar sistemas dinâmicos que apresentam oscilações e caos, biestabilidade e memória, lógica booleana ou comportamentos algorítmicos 30-38. Por exemplo, Ref. 29 demonstraram uma CRN três reacção que fornecida uma implementação molecular de um protocolo de &quot;consenso&quot;, um tipo de algoritmo de computação distribuída 29,39,40. Este trabalho demonstrou pela primeira vez uma nova utilização para o formalismo CRN como uma &quot;linguagem de programação&quot; para sintetizar rapidamente sistemas moleculares funcionais (Figura 1A). Aqui, um protocolo detalhado para derivar portões ndsDNA de ADN de plasmídeo é fornecida. Primeiro é uma revisão do processo de concepção sequência. Depois segue-se uma explicação de como oligonucleótidos sintéticos que contêmas sequências de porta são clonados em plasmídeos e sequência verificada e amplificado através de cultura bacteriana. Em seguida, é mostrado como ndsDNA portas podem ser derivados a partir dos plasmídeos por tratamento enzimático (ver Figura 2). Por fim, um método para testar o comportamento portão usando a cinética de fluorescência os ensaios é descrita. Mecanismo de reacção Como um exemplo, o protocolo centra-se na reacção química catalítica de A + B&gt; B + C. As espécies de A, B, e C (&quot;sinais&quot;, Figura 1B) correspondem todas a uma molécula diferente de cadeia simples de ADN. As sequências destas moléculas são completamente independentes e os fios não reagem um com o outro directamente. As sequências de todos os sinais têm dois domínios funcionais diferentes, ou seja, subsequências que agem em conjunto em reacções de deslocamento de cadeia: 1) um ponto de apoio de domínio curto (etiquetas TA, TB, TC) que é usado para a iniciação da cadeia deslocamento reaction, e 2) um domínio longo (etiquetas a, b, c) que determina a identidade do sinal. Interações entre fios de sinal são mediados por ADN (ndsDNA) Complexos de porta de cadeia dupla com entalhe (chamados Adira AB e Fork BC) e as espécies de cadeia simples auxiliares (&lt;tr r&gt;, &lt;b tr&gt;, &lt;c tb&gt; e &lt;i tc &gt;). A reacção de formal de A + B&gt; B + C é executada através de uma série de passos de reacção de deslocamento de cadeia, em que cada passo reaccional expõe um ponto de apoio para uma reacção subsequente (Figura 1B). Neste exemplo, os sinais A e B são inicialmente livre em solução enquanto o sinal de C está ligado ao portão forquilha. No final da reacção, B e C estão em solução. De modo mais geral, os sinais que estão vinculados a um portão estão inativos, enquanto os sinais que são livres em solução são ativos, isto é, eles podem participar de uma reação de deslocamento de cadeia comouma entrada. O curso do tempo da reacção é seguido usando uma estratégia repórter fluorescente (Figura 1C). Em trabalho anterior 29, demonstrou-se que este mecanismo de reacção, não só realiza a estequiometria correcta, mas também a cinética da reacção alvo.

<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.

Strand DNA Deslocamento Portas para redes de reação de Execução químicos derivados do plasmídeo

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 de Cartagena UDEC

Research

JoVE Journal

Biology

14.3K visualizações.  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. 

Vídeo: 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.

Projetando um robô Bio-responsivo de DNA Origami

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

Bioengenharia

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.

Mutagênese do site caseiros Direção de Plasmids 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.

Robótico líquido manipulação montagem de dispositivos modulares DNA automática

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.

Usando biologia sintética para manipular células vivas que fazem interface com materiais programáveis

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 e síntese de um Rack de acordeão de DNA reconfigurável

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.

Superfície-imobilização funcional dos Genes usando várias etapas Strand deslocamento 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.

Montagem rápida de construções multigênicas usando clonagem modular de 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.

Dna-Tethered RNA Polymerase para Transcrição In vitro Programável e Computação 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 Digitais Gênicos Baseados em Sistemas CRISPR-Cas e 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.

Rato Genoma Engenharia Usando Nucleases Designer

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