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 border="0">
	<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&#160;</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 ...

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JoVE Journal

Biology

14.0K 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

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death. Cells repair DSBs using two major pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). Perturbations of NHEJ and HR are often associated with premature aging and tumorigenesis, hence it is important to have a quantitative way of measuring each DSB repair pathway. Our laboratory has developed fluorescent reporter constructs that allow sensitive and quantitative measurement of NHEJ and HR. The constructs are based on an engineered GFP gene containing recognition sites for a rare-cutting I-SceI endonuclease for induction of DSBs. The starting constructs are GFP negative as the GFP gene is inactivated by an additional exon, or by mutations. Successful repair of the I-SceI-induced breaks by NHEJ or HR restores the functional GFP gene. The number of GFP positive cells counted by flow cytometry provides quantitative measure of NHEJ or HR efficiency.

Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

This article describes GFP-based fluorescence in vivo assays that separately quantify homologous recombination and nonhomologous end joining in mammalian cells.

포유 동물 세포의 DNA 이중 가닥 브레이크 (DSB) 수리 분석

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death.  Cells repair DSBs using ...

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생물학

How to Culture, Record and Stimulate Neuronal Networks on Micro-electrode Arrays (MEAs)

For the last century, many neuroscientists around the world have dedicated their lives to understanding how neuronal networks work and why they stop working in various diseases. Studies have included neuropathological observation, fluorescent microscopy with genetic labeling, and intracellular recording in both dissociated neurons and slice preparations. This protocol discusses another technology, which involves growing dissociated neuronal cultures on micro-electrode arrays (also called multi-electrode arrays, MEAs).
There are multiple advantages to using this system over other technologies. Dissociated neuronal cultures on MEAs provide a simplified model in which network activity can be manipulated with electrical stimulation sequences through the array's multiple electrodes. Because the network is small, the impact of stimulation is limited to observable areas, which is not the case in intact preparations. The cells grow in a monolayer making changes in morphology easy to monitor with various imaging techniques. Finally, cultures on MEAs can survive for over a year in vitro which removes any clear time limitations inherent with other culturing techniques.1
Our lab and others around the globe are utilizing this technology to ask important questions about neuronal networks. The purpose of this protocol is to provide the necessary information for setting up, caring for, recording from and electrically stimulating cultures on MEAs. In vitro networks provide a means for asking physiologically relevant questions at the network and cellular levels leading to a better understanding of brain function and dysfunction.

How to Culture, Record and Stimulate Neuronal Networks on Micro-electrode Arrays MEAs

This protocol provides the necessary information for setting up, caring for, recording from and electrically stimulating cultures on MEAs. In vitro networks provide a means for asking physiologically relevant questions at the network and cellular levels leading to a better understanding of brain function and dysfunction.

어떻게 문화, 기록 및 미세 전극 배열에의 연결을 네트워크를 자극 (MEAs)에

For the last century, many neuroscientists around the world have dedicated their lives to understanding how neuronal networks work and why they stop ...

Bioengineering Human Microvascular Networks in Immunodeficient Mice

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks in vivo. In this regard, the search for experimental models to build blood vessel networks in vivo is of utmost importance 1. The feasibility of bioengineering microvascular networks in vivo was first shown using human tissue-derived mature endothelial cells (ECs) 2-4; however, such autologous endothelial cells present problems for wide clinical use, because they are difficult to obtain in sufficient quantities and require harvesting from existing vasculature. These limitations have instigated the search for other sources of ECs. The identification of endothelial colony-forming cells (ECFCs) in blood presented an opportunity to non-invasively obtain ECs 5-7. We and other authors have shown that adult and cord blood-derived ECFCs have the capacity to form functional vascular networks in vivo 7-11. Importantly, these studies have also shown that to obtain stable and durable vascular networks, ECFCs require co-implantation with perivascular cells. The assay we describe here illustrates this concept: we show how human cord blood-derived ECFCs can be combined with bone marrow-derived mesenchymal stem cells (MSCs) as a single cell suspension in a collagen/fibronectin/fibrinogen gel to form a functional human vascular network within 7 days after implantation into an immunodeficient mouse. The presence of human ECFC-lined lumens containing host erythrocytes can be seen throughout the implants indicating not only the formation (de novo) of a vascular network, but also the development of functional anastomoses with the host circulatory system. This murine model of bioengineered human vascular network is ideally suited for studies on the cellular and molecular mechanisms of human vascular network formation and for the development of strategies to vascularize engineered tissues.

Here, we describe a methodology to deliver human cord blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSCs), embedded in a collagen/fibronectin gel, subcutaneously into immunodeficient mice. This cell/gel combination generates a human vascular network that connects with the mouse vasculature.

Immunodeficient 마우스의 생체 공학 인간 Microvascular 네트워크

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks ...

Iterative Optimization of DNA Duplexes for Crystallization of SeqA-DNA Complexes

Escherichia coli SeqA is a negative regulator of DNA replication that prevents premature reinitiation events by sequestering hemimethylated GATC clusters within the origin of replication1. Beyond the origin, SeqA is found at the replication forks, where it organizes newly replicated DNA into higher ordered structures2. SeqA associates only weakly with single GATC sequences, but it forms high affinity complexes with DNA duplexes containing multiple GATC sites. The minimal functional and structural unit of SeqA is a dimer, thereby explaining the requirement of at least two GATC sequences to form a high-affinity complex with hemimethylated DNA3. Additionally, the SeqA architecture, with the oligomerization and DNA-binding domains separated by a flexible linker, allows binding to GATC repeats separated by up to three helical turns. Therefore, understanding the function of SeqA at a molecular level requires the structural analysis of SeqA bound to multiple GATC sequences. In protein-DNA crystallization, DNA can have none to an exceptional effect on the packing interactions depending on the relative sizes and architecture of the protein and the DNA. If the protein is larger than the DNA or footprints most of the DNA, the crystal packing is primarily mediated by protein-protein interactions. Conversely, when the protein is the same size or smaller than the DNA or it only covers a fraction of the DNA, DNA-DNA and DNA-protein interactions dominate crystal packing. Therefore, crystallization of protein-DNA complexes requires the systematic screening of DNA length4 and DNA ends (blunt or overhang)5-7. In this report, we describe how to design, optimize, purify and crystallize hemimethylated DNA duplexes containing tandem GATC repeats in complex with a dimeric variant of SeqA (SeqA&Delta;(41-59)-A25R) to obtain crystals suitable for structure determination.

Crystal structure of protein&#x2013;DNA complexes can provide insight into protein function, mechanism, as well as, the nature of the specific interaction. Here, we report how to optimize the length, sequence and ends of duplex DNA for co-crystallization with Escherichia coli SeqA, a negative regulator of replication initiation.

SeqA-DNA 단지의 결정에 대한 DNA의 Duplexes의 최적화를 반복

Escherichia coli SeqA is a negative regulator of DNA replication that prevents premature reinitiation events by sequestering hemimethylated GATC clusters ...

Identification of Metabolically Active Bacteria in the Gut of the Generalist Spodoptera littoralis via DNA Stable Isotope Probing Using 13C-Glucose

Guts of most insects are inhabited by complex communities of symbiotic nonpathogenic bacteria. Within such microbial communities it is possible to identify commensal or mutualistic bacteria species. The latter ones, have been observed to serve multiple functions to the insect, i.e. helping in insect reproduction1, boosting the immune response2, pheromone production3, as well as nutrition, including the synthesis of essential amino acids4, among others. &#160; &#160;
Due to the importance of these associations, many efforts have been made to characterize the communities down to the individual members. However, most of these efforts were either based on cultivation methods or relied on the generation of 16S rRNA gene fragments which were sequenced for final identification. Unfortunately, these approaches only identified the bacterial species present in the gut and provided no information on the metabolic activity of the microorganisms.
To characterize the metabolically active bacterial species in the gut of an insect, we used stable isotope probing (SIP) in vivo employing 13C-glucose as a universal substrate. This is a promising culture-free technique that allows the linkage of microbial phylogenies to their particular metabolic activity. This is possible by tracking stable, isotope labeled atoms from substrates into microbial biomarkers, such as DNA and RNA5. The incorporation of 13C isotopes into DNA increases the density of the labeled DNA compared to the unlabeled (12C) one. In the end, the 13C-labeled DNA or RNA is separated by density-gradient ultracentrifugation from the 12C-unlabeled similar one6. Subsequent molecular analysis of the separated nucleic acid isotopomers provides the connection between metabolic activity and identity of the species.
Here, we present the protocol used to characterize the metabolically active bacteria in the gut of a generalist insect (our model system), Spodoptera littoralis (Lepidoptera, Noctuidae). The phylogenetic analysis of the DNA was done using pyrosequencing, which allowed high resolution and precision in the identification of insect gut bacterial community. As main substrate, 13C-labeled glucose was used in the experiments. The substrate was fed to the insects using an artificial diet.

Identification of Metabolically Active Bacteria in the Gut of the Generalist Spodoptera littoralis via DNA Stable Isotope Probing Using 13C-Glucose

The active bacterial community associated with the gut of Spodoptera littoralis, was determined by stable-isotope-probing (SIP) coupled to pyrosequencing. Using this methodology, identification of the metabolically active bacteria species within the community was done with high resolution and precision.

제너럴의 장내 대사 활성 박테리아의 식별<em&gt; 스포 도프 테라 리토 랄</em&gt; DNA 안정 동위 원소를 통해 사용 프로빙<sup&gt; 13</sup&gt; C-포도당

Guts of most insects are inhabited by complex communities of symbiotic nonpathogenic bacteria. Within such microbial communities it is possible to ...

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for expressing proteins and genetic circuits in a controlled manner as well as for providing a prototyping environment for synthetic biology.2,3 To achieve the latter goal, cell-free expression systems that preserve endogenous Escherichia coli transcription-translation mechanisms are able to more accurately reflect in vivo cellular dynamics than those based on T7 RNA polymerase transcription. We describe the preparation and execution of an efficient endogenous E. coli based transcription-translation (TX-TL) cell-free expression system that can produce equivalent amounts of protein as T7-based systems at a 98% cost reduction to similar commercial systems.4,5 The preparation of buffers and crude cell extract are described, as well as the execution of a three tube TX-TL reaction. The entire protocol takes five days to prepare and yields enough material for up to 3000 single reactions in one preparation. Once prepared, each reaction takes under 8 hr from setup to data collection and analysis. Mechanisms of regulation and transcription exogenous to E. coli, such as lac/tet repressors and T7 RNA polymerase, can be supplemented.6 Endogenous properties, such as mRNA and DNA degradation rates, can also be adjusted.7 The TX-TL cell-free expression system has been demonstrated for large-scale circuit assembly, exploring biological phenomena, and expression of proteins under both T7- and endogenous promoters.6,8 Accompanying mathematical models are available.9,10 The resulting system has unique applications in synthetic biology as a prototyping environment, or "TX-TL biomolecular breadboard."

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

This five-day protocol outlines all steps, equipment, and supplemental software necessary for creating and running an efficient endogenous Escherichia coli based TX-TL cell-free expression system from scratch. With reagents, the protocol takes 8 hours or less to setup a reaction, collect, and process data.

구현을위한 프로토콜<em&gt; 대장균</em합성 생물학&gt;을 바탕으로 TX-TL의 무 세포 발현 시스템

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

비례 생체 분자 비콘을 사용하여 살아있는 세포의 단일 엔지니어링 RNA 성적 증명서의 실시간 영상

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Reduced-gravity Environment Hardware Demonstrations of a Prototype Miniaturized Flow Cytometer and Companion Microfluidic Mixing Technology

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If humans are to travel beyond low Earth orbit, a transition towards space-ready, point-of-care (POC) testing is required. Such testing needs to be comprehensive, easy to perform in a reduced-gravity environment, and unaffected by the stresses of launch and spaceflight. Countless POC devices have been developed to mimic laboratory scale counterparts, but most have narrow applications and few have demonstrable use in an in-flight, reduced-gravity environment. In fact, demonstrations of biomedical diagnostics in reduced gravity are limited altogether, making component choice and certain logistical challenges difficult to approach when seeking to test new technology. To help fill the void, we are presenting a modular method for the construction and operation of a prototype blood diagnostic device and its associated parabolic flight test rig that meet the standards for flight-testing onboard a parabolic flight, reduced-gravity aircraft. The method first focuses on rig assembly for in-flight, reduced-gravity testing of a flow cytometer and a companion microfluidic mixing chip. Components are adaptable to other designs and some custom components, such as a microvolume sample loader and the micromixer may be of particular interest. The method then shifts focus to flight preparation, by offering guidelines and suggestions to prepare for a successful flight test with regard to user training, development of a standard operating procedure (SOP), and other issues. Finally, in-flight experimental procedures specific to our demonstrations are described.

Spaceflight blood diagnostics need innovation. Few demonstrations have been published illustrating in-flight, reduced-gravity health diagnostic technology. Here we present a method for construction and operation of a parabolic flight test rig for a prototype point-of-care flow-cytometry design, with components and preparation strategies adaptable to other setups.

프로토 타입 소형 흐름 사이토와 동반자 미세 유체 혼합 기술의 감소 - 중력 환경 하드웨어 데모

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If ...

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of Gi2 (a heterotrimeric G-protein &#945;-subunit) is described in detail.

This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

절대 단백질 정량 질량 분석을 모니터링 선정 반응

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents is important for protecting public health. The in vivo alkaline comet assay, which detects DNA damage as strand breaks, is especially relevant for assessing the genotoxic hazards of xenobiotics, as its responses reflect the in vivo absorption, tissue distribution, metabolism and excretion (ADME) of chemicals, as well as DNA repair process. Compared to other in vivo DNA damage assays, the assay is rapid, sensitive, visual and inexpensive, and, by converting oxidative DNA damage into strand breaks using specific repair enzymes, the assay can measure oxidative DNA damage in an efficient and relatively artifact-free manner. Measurement of DNA damage with the comet assay can be performed using both acute and subchronic toxicology study designs, and by integrating the comet assay with other toxicological assessments, the assay addresses animal welfare requirements by making maximum use of animal resources. Another major advantage of the assays is that they only require a small amount of cells, and the cells do not have to be derived from proliferating cell populations. The assays also can be performed with a variety of human samples obtained from clinically or occupationally exposed individuals.

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

The alkaline comet assay measures DNA strand breaks in eukaryotic cells. By adding an Endonuclease III or human 8-oxoguanine-DNA-N-glycosylase digestion step, the assay can efficiently detect oxidative DNA damage. We describe methods for using these assays to detect DNA damage in rat liver.

쥐 간에서 DNA 가닥 나누기 및 산화 적 DNA 손상을 측정하기위한 생체 알카라인 혜성 분석 및 효소 수정 알카라인 혜성 분석에서

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents ...