Name: plasmid-derived dna strand displacement gates for implementing chemical reaction networks
Uploaded: 2015-11-25T13:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks at JoVE.com

Şekiller 1, 2, 3, 4, 6, 8 ve Tablolar 2, 3, 10, 15, 16 Ref 29 modifiye edilmiştir. Bu çalışma, Ulusal Bilim Vakfı tarafından desteklenmiştir (NSF-CCF 1117143 ve GS NSF-CCF 1162141 hibe). Y.-JC Tayvanlı hükümet Bursu ile desteklenmiştir. SDR Ulusal Bilim Vakfı Lisansüstü Araştırma Bursu Programı (GRFP) tarafından desteklenmiştir.

<ol>
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The authors declare no competing financial interests.

Bu çalışma, yüksek derecede saf plazmid DNA&#39;sından ndsDNA kapıları çıkarmak için bir yöntem tarif eder. Ayrıca, bir protokol bir floresan kinetik analizi kullanılarak kapı performansını karakterize etmek için sunulmuştur. Deneysel veriler, sentetik sistemi poliakrilamid jel elektroforezi (PAGE) ile arıtılmış, şeritlerin monte bile plasmid türevli sistemi sentetik muadili daha iyi performans göstermektedir. Olasılıkla, plazmid kaynaklı kapılarının geliştirilmiş performans biyolojik DNA çok yüksek saflıkta öncelikle kaynaklanmaktadır. Sentetik DNA, tipik olarak bütünüyle PAGE ve yüksek performanslı sıvı kromatografisi (HPLC), saflaştırma prosedürleri kaldırılmaz uzunluğu n-1, ve bu tür yan ürünlerin oligonükleotidlerin neden, özellikle silme hataların çeşitli içerir. Burada bildirilenlere benzer gelişmeler de biyolojik kaynaklardan elde edilen 21 DNA kullanılan katalize firkete yükseltecinin bir önceki çalışmada tespit edilmiştir. <p class = &quot;jove_content&quot;&gt; Ancak, plazmid kaynaklı kapılarının kullanımı bile tamamen en az iki nedeni vardır hangi kapı performans hataları, yok edemez: çok olan kapıları yol açabilir ilk aşırı sindirim ya da kesilmiş hassasiyet eksikliği Yanlış pozisyonlarda çentik veya çentikler. Her iki durumda da, kapılar arzu edilmeyen reaksiyonlara katılma olasılığı daha yüksektir. Bu tür sorunlar, kullanılan enzim miktarı (bakınız Şekil 4) optimize hafifletti edilebilir. İkinci olarak, bu deneylerde, en fazla giriş ve yardımcı şeritler sentetik DNA ve dolayısıyla silmeler ve mutasyonlar ihtiva etmiştir. Prensip olarak, her tek sarmallı giriş ve yardımcı şeritler, ayrıca ön-kodlanmış m13 viral genomun 26 bir çentikleme enzimi sindirimi yoluyla fajemid DNA elde edilebilir. Belki de devresi performansı, bakteriyel genom türetilmiş ssDNA kullanılarak geliştirilebilir. Plasmid türevli kapılarının kullanımı devre performansını geliştirdiği bulunmuştur birlikte, bir analizmaliyet ve üretim koşulları kez ya plasmid türevli kapıların üretimi biraz daha ucuz (Tablo 15) ise, bu ticari olarak sentezlenir oligolar (Tablo 16) için montaj ve kapılar saflaştırılması göre işlem süresi 2-3 kat daha uzun sürer olduğunu ortaya koymuştur. Plasmid türevli kapılarının primer maliyetleri gen sentezi ve sınır enzimleri kullanımı vardır. (30 nM&#39;de 15 reaksiyonlar için yeterli) kapıları 300 pmol için kapıları Üyelik için tahmini maliyeti nedeniyle farklı nicklemek enzimlerin kullanımına yaklaşık 170 $ ve Çatal kapıları, varlık maliyet farkı 200 $ olduğunu. Buna karşılık, bir SAYFA arıtma ücreti dahil 260 $ civarında, aynı kapı masrafları için şeritlerin kimyasal sentez. Plazmid kaynaklı kapıları için birincil saat maliyeti sadece DNA sentezi gibi, bir gen sentezi şirkete taşeron olabilir, klonlama prosedürü yer almaktadır. Bununla birlikte, bir kez monte plasmid türevli kapıları konakçı plasmidler kolayca çoğaltılabilir avantajınd bakteriyel gliserol stokları halinde saklanabilir. Bu mümkün üzerinde Gates birçok kez yeniden yapar. İleriye yönelik olarak, plazmid kaynaklı kapılarının iyileştirilmiş performans deneysel kadar DNA CRNs ile ortaya konmuştur daha dinamik davranışları çok daha büyük bir yelpazede sağlayabilir. Örneğin, son teorik çalışma 47,48 makro ölçekte kendi kendine organize mekansal desen bir reaksiyon difüzyon mekanizması aracılığıyla DNA CRNs ile gerçekleştirilebilir önerdi. Burada sunulan yöntem, öz-desenlendirme DNA malzemeler için altta yatan moleküler bileşenleri oluşturmak için uygun bir yol sağlar. Zorlu olsa da, programlanabilir bir şekilde makro ölçekli morfolojileri gelişmekte biyomalzemeler araştırma rejeneratif tıp kadar alanlarda önemli etkileri olacaktır.

Watson-Crick baz eşleşmesi öngörülebilirliği dinamik DNA nanoteknoloji dinamik özelliklerinin 1,2 ile moleküler cihazlar tasarlamak için programlanabilir bir yolu olarak ortaya sağladı. Özellikle, DNA zinciri değiştirme - programlanabilir, rekabetçi melezleme tepki - Dinamik DNA sistemleri mühendisliği için güçlü bir mekanizma olduğunu kanıtlamıştır. Bir DNA sarmal yer değiştirme reaksiyonunda, gelen bir oligonükleotid, bir tamamlayıcı bağlama ortağı ile ilgili daha önce bağlanmış &quot;Çıkış&quot; strand değiştirir. Birden tür reaksiyonlar düzen ve bireysel tepki zamanlaması 3. adımları üzerinde kontrol yüksek derecede çok adımlı reaksiyon kaskadları halinde bir araya zincirleme olabilir. DNA zincir değiştirme kaskadları dijital ve analog devreler moleküler 4-7, değiştirilebilir nano 8-10, özerk moleküler motorlar 11-15 ve non-kovalent katalitik yükselticiler 13,16-21 oluşturmak için kullanılmıştır. Ayrıca, DŞerit değiştirme reaksiyonları kullanılarak NA cihazlar simüle ve bilgisayar destekli tasarım yazılımı kullanılarak 22-24 farklı uygulamalar için dizayn edilebilir. Şu anda, kimyasal olarak sentezlenmiş DNA, DNA nanoteknoloji ana malzeme olarak işlev görür. Bununla birlikte, DNA sentezi işleminde hatalar ve elde edilen kusurlu oligonükleotitler, yanlış bir yan reaksiyonların neden olarak, dinamik bir DNA cihazların performansı sınırladığına inanılır. Örneğin, &quot;kaçak&quot; reaksiyonlar, hatta bir reaksiyon tetikleyici yokluğunda bir çıkış oligonükleotidin serbest bırakılması ile sonuçlanabilir. Bu etkiler ilk sızıntı bile az miktarda sonunda çağlayan 19,20 tam aktivasyonuna neden olur otokatalitik tepki kaskadları en ortada. Tersine, tepkiler genellikle bazı bileşenler bile amaçlanan giriş 7,25 varlığında tetiklemek çünkü aktivasyon beklenen seviyeye ulaşmak için başarısız. Performansını yapmak için DNA temellibiyolojik protein bazlı muadillerine karşılaştırılabilir nanoaygıtlar, böyle bir hata modları önemli ölçüde azalır gerekmektedir. Bakteriyel plazmidler veya diğer biyolojik DNA nanoteknoloji uygulamaları için, son derece saf bir DNA nispeten ucuz kaynağı olarak hizmet edebilir. DNA&#39;nın büyük miktarlarda bakteri de kopyalama ile üretilmiş olabilir ve canlı sistemlerin içsel düzeltme okuması özellikler elde edilen DNA saflığı sağlamak. Aslında, birçok yeni belgeler nanoteknoloji uygulamaları 21,26-28 biyolojik DNA potansiyel yarar tanıdı. Bununla birlikte, plasmid DNA, tam çift sarmal yapısı şimdiye kadar, tipik olarak çok sayıda oligonükleotitlerin oluşur ve Çift dallı ve tek dallı domeynini içeren dinamik bir DNA aygıtları yapmak için bir malzeme olarak kullanımı yasaklamıştır. Son kağıdı 29 bu konu ele alındı ​​ve öncelikle çentikli çift iplikli DNA (ndsDNA) oluşan yeni bir DNA kapı mimarisinde tanıtmak oldud. Önemlisi, ndsDNA kapıları sistemleri herhangi bir resmi kimyasal reaksiyon ağı (CRN) 29 tarafından belirlenen dinamikleri fark etmesi dizayn edilebilir. ndsDNA kapıları böylece salınımları ve kaos, İki sabit ve hafıza, Boole mantığı veya algoritmik davranışlar sergilemek 30-38 dinamik sistemler oluşturmak için prensipte kullanılabilir. Örneğin, Ref. 29, bir &quot;konsensüs&quot; protokol bir molekül uygulanması, dağıtık bilgisayar algoritması 29,39,40 bir tür mesafede üç Reaksiyon CRN gösterdi. Bu çalışma ilk olarak hızla fonksiyonel moleküler sistemleri (Şekil 1A) sentezlemek için bir &quot;programlama dili&quot; olarak CRN biçimcilik için yeni bir kullanım göstermiştir. Burada, plasmid DNA ndsDNA kapıları türetmek için detaylı bir protokol verilmektedir. İlk dizi tasarım sürecinin bir yorum olduğunu. Daha sonra içeren ne kadar, sentetik oligonükleotidlerin bir açıklama aşağıdaKapı dizileri plasmidler klonlanır ve sekansı doğrulanır ve bakteri kültürü ile amplifiye edilmiştir. NdsDNA kapılar, enzimatik işlemle plazmidlerden türetilebilir kadar Daha sonra, gösterilir (bakınız Şekil 2). Son olarak, flüoresan kinetik analizleri kullanılarak kapı davranışı test etmek için bir yöntem, ana hatlarıyla verilmiştir. Reaksiyon mekanizması Bir örnek olarak, iletişim kuralı, katalitik kimyasal reaksiyon A + B-&gt; B + C yöneliktir. Türleri A, B, ve C (&quot;sinyalleri&quot;, Şekil 1B) her biri farklı bir tek sarmallı bir DNA molekülüne karşılık gelir. Bu moleküllerin dizileri tamamen bağımsızdır ve şeritler birbirleriyle doğrudan reaksiyona girmeyen. Tüm sinyaller sekansları, yani, iki farklı fonksiyonel alanları, sarmal yer değiştirme reaksiyonlarında birlikte hareket dizilerine sahiptir: 1) kısa bir toehold alanı (etiketler sarmal yer değiştirme r başlatılması için kullanılır, tb, tc) taeaction ve 2) uzun bir etki (a, b, c) sinyal kimliğini belirleyen etiketler. Sinyal teller arasındaki etkileşimler tırtıklı çift iplikli DNA (ndsDNA) kapı kompleksleriyle ve yardımcı tek sarmallı türler (&lt;tr r&gt;, &lt;tb c&gt; ve &lt;tr b&gt; &lt;i tc (denilen AB ve Çatal BC Katıl) aracılık &gt;). + B-&gt; B + C formal Reaksiyon bir biçimde her bir reaksiyon aşaması, bir sonraki reaksiyon (Şekil 1B) bir toehold ortaya sarmal yer değiştirme reaksiyon aşamalarının, bir dizi yürütülür. Sinyal Cı çatal geçidine bağlıdır Bu örnekte, A ve B sinyalleri ilk solüsyonda serbest olan. Reaksiyon B ve C sonunda çözelti içinde bulunmaktadır. Daha genel olarak, bir kapı bağlı olan sinyaller solüsyonda serbest olan sinyaller etkin iken, yani, bir sarmal yer değiştirme reaksiyonu olarak katılabilir inaktiftirBir giriş. Reaksiyon süresi programı bir flüoresan raportör stratejisi (Şekil 1C) ile takip edilir. Önceki çalışmaları 29, bu reaksiyon mekanizmaları doğru stoikiometriyi yanı sıra hedef reaksiyon kinetiklerini fark sadece bu gösterilmiştir.

<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.

Plazmid kökenli Uygulama Kimyasal Reaksiyon Ağları için DNA Strand Deplasman kapıları

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

Nanyang Technological University

Research

JoVE Journal

Biology

14.3K Görüntüleme.  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.

DNA Origami bir Bio-duyarlı Robot Tasarımı

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

Biyomühendislik

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.

Tüm Plazmidler Homemade Sitesi Yönetmen Mutagenez

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

Biyoloji

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.

Robot sıvı işleme derleme modüler DNA cihazların otomatik

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.

Yaşayan Hücreleri Mühendise Sentetik Biyoloji kullanarak That Programlanabilir Malzemelerle Arayüzü

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.

Tasarım ve Reconfigurable DNA akordeon raf sentezi

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.

Genlerin Multistep Strand deplasman litografi kullanarak işlevsel yüzey immobilizasyon

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.

Modüler Altın Kapı Klonlama Kullanılarak Çok Genli Yapıların Hızlı Montajı

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.

Programlanabilir In vitro Transkripsiyon ve Moleküler Hesaplama için DNA-Bağlı RNA Polimeraz

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.

CRISPR-Cas Sistemlerine ve Anti-CRISPR Proteinlerine Dayalı Gen Dijital Devreleri

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.

Plazmid vektörler oluşturulması Gibson tertibatı kullanılarak insan uzama faktör-1α bir promotörün regülasyonu altında FLAG-etiketli Proteinlerinin İfade

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