기금은 고든 무어와 베티 재단 보조금 및 국립 과학 재단 (National Science Foundation) 부여 MCB - 0702125에 의해 제공되었다.

<ol>
	<li>Liang, P., Pardee, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Science. 257 (5072), 967-967 (1992).</li><li>Belasco, J. G., Belasco, J. G., Brawerman, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+messenger+RNA+stability.">Control of messenger RNA stability.</a> , 3-11 (1993).</li><li>Poretsky, R. S., Bano, N., Buchan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 71 (7), 4121-4121 (2005).</li><li>Gelder, R. N. V., von Zastrow, M. E., Yool, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Natl. Acad. Sci. USA. 87 (5), 1663-1663 (1990).</li><li>Margulies, M., Egholm, M., Altman, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature. 437 (7057), 376-376 (2005).</li><li>Frias-Lopez, J., Shi, Y., Tyson, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Natl. Acad. Sci. USA. 105 (10), 3805-3805 (2008).</li><li>Poretsky, R. S., Hewson, I., Sun, S., Allen, A. E., Zehr, J. P., Moran, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Environ Microbiol. 11 (6), 1358-1375 (2009).</li></ol>

천연 미생물 커뮤니티에 의해 유전자 발현의 조사 미생물의 생태 역할과 기능을 탐구하는 수단으로 최근 몇 년 동안 일반되었다. 해양 미생물의 감지, 부량, 그리고 특성화와 함께 사용, 유전자 발현의 분석은 자연 미생물 지역 사회에 기능 phylogeny를 연결하는 데 사용할 수 있습니다. 기능성 유전자 발현을 대상으로 많은 기술은 특정 프로브 또는 알려진 일련의 유전자위한 프라이머 세트에 의존?...

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<tr>
<td>PowerSoil Bead Tubes</td>
<td>kit</td>
<td>MoBio</td>
<td>12866-25-PBT</td>
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<td>RNeasy Mini Kit</td>
<td>kit</td>
<td>QIAGEN</td>
<td>74104</td>
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<td>TURBO DNA-free</td>
<td>kit</td>
<td>Ambion</td>
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<td>mRNA-ONLY Prokaryotic mRNA Isolation Kit</td>
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<td>kit</td>
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<td>MICROBEnrich kit</td>
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<td>Universal RiboClone cDNA Synthesis System</td>
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<td>Wizard DNA Clean-Up System</td>
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analyzing gene expression from marine microbial communities using environmental transcriptomics

metagenomics 유사, 환경 transcriptomics (metatranscriptomics) 검색 및 커뮤니티 표현이 될 일을 유전자의 사전 지식없이도 미생물의 조립에서 시퀀스 환경 mRNAs합니다. 그러므로 그것은 사회의 유전자 발현에 가장 불편 관점을 제공합니다<em&gt; 현장에서</em&gt;. prokaryotic mRNAs은 일반적으로 비교적 간단 진핵세포 메시지의 고립을 폴리 (A) 꼬리가 부족 때문에 환경 transcriptomics 프로토콜은 기술적으로 어렵습니다<sup&gt; 1</sup&gt; 때문에 mRNAs의 비교적 짧은 반 삶의<sup&gt; 2</sup&gt;. 또한, mRNAs 훨씬 덜 풍부한 총 RNA 추출물에 rRNAs보다되며, 따라서 rRNA 배경은 종종 mRNA 신호를 압도. 그러나, 이러한 어려움의 일부를 극복을위한 기술은 최근에 개발되었습니다. 리버스 녹음과 환경 mRNAs를 증폭하기 위해 무작위 primers를 사용하여 복제 라이브러리를 작성하여 환경 transcriptomes 분석을위한 절차는 최근에 두 개의 서로 다른 자연 환경에서 성공적으로 설명했지만, 결과는 cDNA 합성을 시작하는 데 사용되는 임의 primers의 선택에 의해 편향된되었습니다<sup&gt; 3</sup&gt;. mRNA의 선형 증폭의 진보는 증폭 단계에서 무작위로 primers의 필요성을 사전에 제거하다 그것이 가능한 샘플의 수집과 처리 시간을 감소시키고 RNA 저하를 최소화 적은 시작 자료를 사용할 수 있도록<sup&gt; 4</sup&gt;.<em&gt; 체외에서</em확장 mRNA에 대한&gt; 전송 방법은 mRNA를 polyadenylating 및 성적 증명서의 3 월말에 T7 프로 모터를 통합하는 방식입니다. 증폭된 RNA (아르나)는 pyrosequencing하여 다음 임의 hexamers를 사용하여 cDNA를 두 번 좌초로 변환하고 직접 합성 수 있습니다<sup&gt; 5</sup&gt;. 역 알로하에서이 메서드의 첫 번째 사용은 미생물 사회의 유전자 발현을 특성화에 대한 유틸리티를 시연<sup&gt; 6</sup&gt;.

Watch this Scientific Journal Video about Analyzing Gene Expression from Marine Microbial Communities using Environmental Transcriptomics at JoVE.com

Analyzing Gene Expression from Marine Microbial Communities using Environmental Transcriptomics

우리는 환경 mRNA로부터 cDNA 생성하는 방법을 제시한다. 일반적으로, 총 RNA는 먼저 환경에서 수집, rRNA는 선택, 제거 mRNA가 선택적으로 증폭되고 농축 mRNA 수영장에서 합성된 cDNA가 합성됩니다. 복구된 시퀀스는 표현 유전자를 식별하는 표준 생물 정보학 기술을 사용하여 주석하실 수 있습니다.

환경 Transcriptomics를 사용하여 해양 미생물 커뮤니티의 유전자 발현 분석

Analogous to metagenomics, environmental transcriptomics (metatranscriptomics) retrieves and sequences environmental mRNAs from a microbial assemblage ...

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Research

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12.4K 조회수.  University of Georgia (UGA). 우리는 환경 mRNA로부터 cDNA 생성하는 방법을 제시한다. 일반적으로, 총 RNA는 먼저 환경에서 수집, rRNA는 선택, 제거 mRNA가 선택적으로 증폭되고 농축 mRNA 수영장에서 합성된 cDNA가 합성됩니다. 복구된 시퀀스는 표현 유전자를 식별하는 표준 생물 정보학 기술을 사용하여 주석하실 수 있습니다.

비디오: Analyzing Gene Expression from Marine Microbial Communities using Environmental Transcriptomics

Microbial Communities in Nature and Laboratory - Interview

자연과 실험실에서 미생물 커뮤니티 - 인터뷰

연구

생물학

Biology of Microbial Communities  - Interview

미생물 커뮤니티의 생물학 - 인터뷰

Bacterial Gene Expression Analysis Using Microarrays

Microarrays을 사용하여 세균의 유전자 발현 분석

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

Electroporation에 대한 Murine 골수에서 조혈 세포 차 문화의 준비

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

고효율 기본 셀을 Transfect하는 진 Pulser Electroporation MXcell의 시스템을 사용하여

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

유전자 발현 연구를 단순화하는 자동 셀 카운터를 사용하여 : Namalwa 셀에서 IL - 4 종속 유전자 발현의 siRNA의 최저

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

활성 T.의 체외의 Reconstitution에서 castaneum의 Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Extraction of High Molecular Weight DNA from Microbial Mats

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction of high-quality, high molecular weight (HMW) community DNA. However, environmental mat samples often pose difficulties to obtaining large concentrations of high-quality, HMW DNA. Hypersaline microbial mats contain high amounts of extracellular polymeric substances (EPS)1 and salts that may inhibit downstream applications of extracted DNA. Direct and harsh methods are often used in DNA extraction from refractory samples. These methods are typically used because the EPS in mats, an adhesive matrix, binds DNA2,3 during direct lysis. As a result of harsher extraction methods, DNA becomes fragmented into small sizes4,5,6.
The DNA thus becomes inappropriate for large-insert vector cloning. In order to circumvent these limitations, we report an improved methodology to extract HMW DNA of good quality and quantity from hypersaline microbial mats. We employed an indirect method involving the separation of microbial cells from the background mat matrix through blending and differential centrifugation. A combination of mechanical and chemical procedures was used to extract and purify DNA from the extracted microbial cells. Our protocol yields approximately 2 &mu;g of HMW DNA (35-50 kb) per gram of mat sample, with an A260/280 ratio of 1.6. Furthermore, amplification of 16S rRNA genes7 suggests that the protocol is able to minimize or eliminate any inhibitory effects of contaminants. Our results provide an appropriate methodology for the extraction of HMW DNA from microbial mats for functional metagenomic studies and may be applicable to other environmental samples from which DNA extraction is challenging.

We provide an improved protocol for extracting high molecular weight DNA from hypersaline microbial mats. Microbial cells are separated from the mat matrix prior to DNA extraction and purification. This enhances the concentrations, quality, and size of the DNA. The protocol may be used for other refractory samples.

미생물 매트에서 높은 분자량의 DNA의 추출

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction  of high-quality, high molecular weight ...

Concentration of Metabolites from Low-density Planktonic Communities for Environmental Metabolomics using Nuclear Magnetic Resonance Spectroscopy

Environmental metabolomics is an emerging field that is promoting new understanding in how organisms respond to and interact with the environment and each other at the biochemical level1. Nuclear magnetic resonance (NMR) spectroscopy is one of several technologies, including gas chromatography&#x2013;mass spectrometry (GC-MS), with considerable promise for such studies. Advantages of NMR are that it is suitable for untargeted analyses, provides structural information and spectra can be queried in quantitative and statistical manners against recently available databases of individual metabolite spectra2,3. In addition, NMR spectral data can be combined with data from other omics levels (e.g. transcriptomics, genomics) to provide a more comprehensive understanding of the physiological responses of taxa to each other and the environment4,5,6. However, NMR is less sensitive than other metabolomic techniques, making it difficult to apply to natural microbial systems where sample populations can be low-density and metabolite concentrations low compared to metabolites from well-defined and readily extractable sources such as whole tissues, biofluids or cell-cultures. Consequently, the few direct environmental metabolomic studies of microbes performed to date have been limited to culture-based or easily defined high-density ecosystems such as host-symbiont systems, constructed co-cultures or manipulations of the gut environment where stable isotope labeling can be additionally used to enhance NMR signals7,8,9,10,11,12. Methods that facilitate the concentration and collection of environmental metabolites at concentrations suitable for NMR are lacking. Since recent attention has been given to the environmental metabolomics of organisms within the aquatic environment, where much of the energy and material flow is mediated by the planktonic community13,14, we have developed a method for the concentration and extraction of whole-community metabolites from planktonic microbial systems by filtration. Commercially available hydrophilic poly-1,1-difluoroethene (PVDF) filters are specially treated to completely remove extractables, which can otherwise appear as contaminants in subsequent analyses. These treated filters are then used to filter environmental or experimental samples of interest. Filters containing the wet sample material are lyophilized and aqueous-soluble metabolites are extracted directly for conventional NMR spectroscopy using a standardized potassium phosphate extraction buffer2. Data derived from these methods can be analyzed statistically to identify meaningful patterns, or integrated with other omics levels for comprehensive understanding of community and ecosystem function.

A method for metabolite extraction from microbial planktonic communities is presented. Whole community sampling is achieved by filtration onto specially prepared filters. After lyophilization, aqueous-soluble metabolites are extracted. This approach allows for application of environmental metabolomics to trans-omics investigations of natural or experimental microbial communities.

핵 자기 공명 분광학을 사용하여 환경 Metabolomics를위한 낮은 밀도 Planktonic 커뮤니티에서 Metabolites의 농도

Environmental metabolomics is an emerging field that is promoting new understanding in how organisms respond to and interact with the environment and each ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Ribosome 바운드 Polypeptides을 파악하는 도구로 SecM 체포 시퀀스를 사용하여

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...