Name: plasmid-derived dna strand displacement gates for implementing chemical reaction networks
Uploaded: 2015-11-25T13:00:00.000+00:00
Duration: 7 min 50 s
Description: Watch this Scientific Journal Video about Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks at JoVE.com

도 1, 2, 3, 4, 6, 8, 표 2, 3, 10, 15, 16은 참조 번호 (29)에서 수정된다. 이 작품은 국립 과학 재단 (National Science Foundation)에 의해 지원되었다 (NSF-CCF 1,117,143 및 GS에 NSF-CCF 1162141을 부여). Y.-JC는 대만 정부 동호회에 의해 지원되었다. SDR은 국립 과학 재단 (National Science Foundation) 대학원 연구 활동 프로그램 (GRFP)에 의해 지원되었다.

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The authors declare no competing financial interests.

이 논문은 고순도의 플라스미드 DNA에서 ndsDNA 게이트를 유도하는 방법을 설명합니다. 또한, 프로토콜은 형광 동력학 분석을 이용하여 게이트의 성능을 특징으로 제시된다. 실험 데이터는 합성 시스템은 폴리 아크릴 아미드 겔 전기 영동 (PAGE)을 이용하여 정제 가닥 조립 되더라도 플라스미드 유래 된 시스템의 성능을 능가 합성 대응을 보여준다. 가능성, 플라스미드 유래 게이트의 향상된 성능은 생물학적 DNA의 매우 높은 순도에 주로 기인한다. 합성 DNA는 일반적으로 완전히 PAGE 또는 고성능 액체 크로마토 그래피 (HPLC) 정제 과정에서 제거되지 않은 길이 n-1, 및 부 생성물의 발생, 특히 올리고 뉴클레오티드의 결실 다양한 오류를 포함한다. 여기에보고 된 것과 유사한 개선은 또한 생물학적 소스 (21)로부터 유래 된 DNA를 사용하는 촉매 헤어핀 증폭기의 이전 연구에서 관찰되었다. <p cl엉덩이 = &quot;jove_content&quot;&gt; 그러나, 플라스미드 유래의 게이트에도 사용이 완전히 적어도 두 가지 이유가있는 게이트 성능 오차 제거 할 수 너무 많은와 게이트에 이어질 수있는 제 오버 분해 또는 절단 정밀도 부족 잘못된 위치에 흠이나 흠. 두 경우 모두, 게이트는 바람직하지 않은 반응에 참여할 가능성이 높습니다. 이러한 문제점은 사용 된 효소의 양 (도 4 참조)의 최적화에 의해 완화 될 수있다. 둘째,이 실험에서 대부분의 입력과 보조 가닥 합성 DNA이었다 따라서 삭제 및 돌연변이가 포함되어 있습니다. 원칙적으로, 모든 단일 가닥 및 보조 입력 스트랜드는 또한 사전 부호화 M13 바이러스 게놈 (26)의 칼자국을내는 효소 절단을 통해 파지 미드 DNA로부터 수득 될 수있다. 아마도 회로 성능은 또한 세균 유래의 게놈을 ssDNA를 사용하여 향상시킬 수있다. 플라스미드 유래 게이트의 사용은 회로 성능을 개선하는 것으로 확인되었지만, 분석비용과 처리 시간의 플라스미드 유래 게이트의 생산이 다소 저렴 (표 15)이지만, 상업적으로 합성 올리고 (표 16)에서 조립 및 게이트의 정화에 비해 처리 시간이 2 ~ 3 배 더 오래 걸립니다 것으로 나타났다. 플라스미드 유래 게이트의 기본 비용 유전자 합성 및 제한 효소의 사용이다. (30 nm에서 15 반응에 대한 충분한) 게이트의 300 pmole의 경우, 게이트에 참여하기위한 예상 비용 때문에 다른 새김 효소의 사용으로 약 $ 170, 포크 게이트되는 비용 차이에 대한 $ 200. 대조적으로, PAGE 정제 비용을 포함해서 약 $ 260 같은 게이트 비용 가닥의 화학적 합성. 플라스미드 유래 게이트 용 기본 시간 비용은 DNA 합성과 마찬가지로 합성 유전자 회사에 아웃소싱 될 수 있고, 클로닝 절차이다. 그러나, 한번 조립 플라스미드 유래의 게이트는 숙주 플라스미드 쉽게 복제 될 수있는 이점을 가지고ND 세균성 글리세롤 주식의 형태로 저장 될 수있다. 이로써 위에 게이트 여러 번 재사용 할 수있다. 기대, 플라스미드 유래 게이트의 향상된 성능을 실험적으로 지금까지 DNA CRNs으로 입증 된 것보다 다이나믹 행동의 훨씬 더 큰 범위를 가능하게 할 수있다. 예를 들어, 최근의 이론적 워크 47, 48은 매크로 스케일에서 자기 조직화 공간 패턴이 반응 확산 장치를 통해 DNA CRNs로 실현 될 수 있음을 제안 하였다. 여기에 제시된 방법은 자기 패터닝 재료 용 DNA 분자 기본 요소를 구성하기위한 가능한 경로를 제공한다. 도전하지만, 프로그래밍 방식으로 매크로 규모의 형태학을 개발하는 것은 생체 재료 연구에서 재생 의학에 이르기까지 다양한 분야에서 상당한 영향을 미칠 것입니다.

왓슨 - 크릭 염기 쌍의 예측은 동적 DNA 나노 기술은 동적 속성 1, 2와 분자 장치를 설계하는 프로그램 방법으로 등장 할 수있다. 특히, DNA 가닥 변위 - 프로그램, 경쟁력 혼성화 반응 - 동적 DNA 시스템 엔지니어링을위한 강력한기구 인 것을 입증했다. DNA의 나선 치환 반응에서, 들어오는 올리고 뉴클레오티드 상보 적 결합 파트너의 결합 이전에 &quot;출력&quot;가닥 변위. 다수의 이러한 반응은 반응 순서 및 개개의 타이밍을 3 단계에 걸쳐 고도의 제어와 다단계 반응 캐스케이드로 서로 연결된다. DNA 나선 치환 캐스케이드은 디지털 및 아날로그 회로 분자 4-7, 8-10 나노 전환, 자율 분자 모터 11-15 및 비공유 증폭기 13,16-21 촉매를 생성하는데 사용되어왔다. 또한, D가닥 치환 반응을 사용하여 NA 디바이스 시뮬레이션 및 컴퓨터 보조 설계 소프트웨어를 사용하여 22-24의 다양한 애플리케이션을 위해 설계 될 수있다. 현재, 화학​​적으로 합성 된 DNA는 DNA 나노 기술의 주요 재료로 작용한다. 그러나, DNA 합성 과정에서 오류 및 결과 불완전한 올리고 뉴클레오티드, 잘못된 부반응시킴으로써 DNA 동적 장치의 성능을 제한하는 것으로 여겨진다. 예를 들어, &quot;누설&quot;반응에도 반응 트리거 부재 출력 올리고 뉴클레오타이드의 방출을 초래할 수있다. 이러한 효과는 초기 누출의 경우에도 최소한 결국 캐스케이드 (19, 20)의 전체 활성화가 발생합니다 촉매 반응 계곡에서 가장 명백하다. 반대로, 반응은 종종 일부 구성도 의도 입력 7,25의 존재하에 유발하지 않기 때문에 활성화 기대 수준에 도달하지 못한다. 의 성능을 확인하기 위해 DNA를 기반생물학적 단백질 기반 대상에 필적 나노 소자는, 예컨대 에러 모드 대폭 저감 할 필요가있다. 세균의 플라스미드 또는 다른 생물학적 DNA는 나노 기술 응용 프로그램을위한 고순도의 DNA의 상대적으로 저렴한 소스의 역할을 할 수있다. 많은 양의 DNA가 박테리아에서 복제함으로써 생성 될 수 있고, 생명체의 극한 교정 능력 얻어진 DNA의 순도를 보장한다. 사실, 몇 가지 최근의 논문은 나노 기술 응용 21,26-28에 대한 생물학적 DNA의 잠재적 인 유틸리티를 인정했다. 그러나, 플라스미드 DNA의 완전 이중 가닥 특성은 지금까지 일반적으로 다수의 올리고 뉴클레오티드로 구성되며, 이중 가닥과 단일 가닥 영역 모두를 포함하는 DNA 동적 장치를 제조하기위한 재료로서 사용을 금지하고있다. 최근 종이 (29)이 문제가 해결되었고, 주로 흠 이중 가닥 DNA (ndsDNA)로 구성되어 새로운 DNA 게이트 구조에서 소개했다디. 중요한 ndsDNA 게이트 시스템은 임의의 형식적인 화학 반응 네트워크 (CRN) (29)에 의해 지정된 역학을 실현하는 설계 될 수있다. ndsDNA 게이트 따라서 진동과 혼란, 쌍 안정성 및 메모리, 부울 논리 또는 알고리즘 행동 30-38을 나타내는 동적 시스템을 만들 원칙적으로 사용될 수있다. 예를 들어, 참조 번호. 29 &quot;합의&quot;프로토콜의 구현 분자량, 분산 컴퓨팅 29,39,40 알고리즘의 타입을 제공하는 세 CRN 반응을 보여 주었다. 이 작품은 처음 빠르게 기능 분자 시스템을 (그림 1A)를 합성하는 &quot;프로그래밍 언어&quot;로 CRN의 형식주의에 대한 새로운 사용을 보여 주었다. 여기서, 플라스미드 DNA에서 ndsDNA 게이트를 유도 상세한 프로토콜이 제공된다. 먼저 시퀀스 설계 프로세스를 검토한다. 그런 다음이 포함 방법 합성 올리고 뉴클레오티드에 대한 설명을 다음과게이트 서열은 플라스미드에 클로닝하고 서열을 확인 및 세균 배양을 통해 증폭. ndsDNA 게이트가 효소 처리에 의해 플라스미드로부터 유도 될 수있는 방법 다음에,이 도시되어있다 (도 2 참조). 마지막으로, 형광 역학 분석을 이용하여 게이트 작동을 테스트하는 방법이 요약되어있다. 반응 메커니즘 예로서, 프로토콜은 촉매 화학 반응 A + B-&gt; B + C에 초점을 맞춘다. 종 A, B, 및 C ( &quot;신호&quot;,도 1b)가 다른 모든 단일 가닥 DNA 분자에 대응한다. 이들 분자의 서열과 완전히 무관 한 가닥은 서로 직접 반응하지 않는다. 모든 신호들의 시퀀스, 즉 두 개의 다른 기능 영역, 나선 치환 반응에서 함께 작용 시퀀스 : 1)으로 발 짧은 도메인 (나선 치환 라벨 (R)의 개시를 위해 사용되며, TB, TC) TAeaction, 2) 긴 도메인 (를 A, B, C) 즉, 신호 식별을 결정 라벨. 신호 가닥 사이의 상호 작용은 흠 이중 가닥 DNA (ndsDNA) 게이트 단지로 및 보조 단일 가닥 종 (&lt;TR R&gt;, &lt;TB C&gt;와 &lt;TR B&gt; &lt;I TC (라고 AB와 포크 기원전 가입) 매개되는 &gt;). + B-&gt; B + C 공식 반응은 각각의 반응 단계가 후속 반응 (도 1b) 용으로 발을 노출 나선 치환 반응 단계들의 시리즈를 통해 실행된다. 신호 C는 포크 게이트에 결합 된 상태에서이 예에서, 신호 A와 B는 초기 용액에 자유 롭다. 반응 B와 C의 끝에 용액이다. 보다 일반적으로, 게이트에 결합되는 신호들은 용액에서 자유롭게 신호가 활성화되어있는 동안, 즉, 그들이 나선 치환 반응에 참여할 수있는 등의 비활성입력. 반응 시간 코스는 형광 리포터 전략 (도 1C)를 사용하여 이어진다. 이전의 연구 (29)에,이 반응 메커니즘은 정확한 화학량뿐만 아니라 대상의 반응 속도론을 행하게하는 것이 입증되었다.

<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.

플라스미드 유래 구현 화학 반응 네트워크 용 DNA 가닥 변위 게이츠

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

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

Universidad San Sebastian

Research

JoVE Journal

Biology

14.3K 조회수.  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. 

비디오: 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 종이 접기에서 바이오 반응 로봇을 설계

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

연구

생체공학

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.

전체 Plasmids의 수제 사이트 감독 돌연변이 유발

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

생물학

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.

모듈형 DNA 장치 조립 로봇 액체 처리 자동화

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.

살아있는 세포를 엔지니어하는 합성 생물학을 사용하여 해당 프로그램 자료와 인터페이스

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.

재구성 가능한 DNA 아코디언 랙의 설계 및 합성

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.

다단계 가닥 변위 리소 그래피를 사용 하 여 유전자의 기능 표면-동원 정지

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.

모듈형 골든 게이트 복제를 사용하여 다중 유전자 구조물의 신속한 조립

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.

시험관 내 전사 및 분자 계산을 위한 DNA 테더드 RNA 폴리머라제

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.

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.

플라스미드 벡터를 생성 깁슨 어셈블리를 사용하여 인간의 신장 요인-1α의 발기인의 규정에 따라 FLAG-태그 단백질을 표현

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