Name: dna-affinity-purified chip (dap-chip) method to determine gene targets for bacterial two component regulatory systems
Uploaded: 2014-07-21T14:00:00.000+00:00
Duration: 12 min 24 s
Description: Watch this Scientific Journal Video about DNA-affinity-purified Chip DAP-chip Method to Determine Gene Targets for Bacterial Two component Regulatory Systems at JoVE.com

우리는 비디오 촬영을 위해 준비하는 그녀의 도움과 기술을 보여 에이미 첸 감사합니다. 수수께끼에 의해 수행이 작품 : 유전자 및 분자 조립 (http://enigma.lbl.gov)와 생태계 및 네트워크 통합은, 로렌스 버클리 국립 연구소의 과학적인 초점 영역 프로그램은 과학, 생물의 사무실의 사무실에 의해 지원되었다 계약 번호 DE-AC02-05CH11231에서 미국 에너지 국의 환경 연구.

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저자는 공개 할 관심의 충돌이 없습니다.

여기에 설명 된 칩 DAP 방법은 성공적 일이 대표적인 결과로 게재되어있는 Desulfovibrio 불가리스 Hildenborough 7 몇개의 RR 대한 유전자 표적을 결정하는 데 사용되었다. RR의 DVU3023 들어, 후보 유전자 표적을 선택하는 것은 수월했다. DVU3025은 RR 유전자의 바로 하류에 위치하고 있으며, RR 및 대상 유전자는 여러 Desulfovibrio 종에서 보존하고, 또한 DVU3025는 예측 sigma54 의존 발기인 있습?...

생존하고 번성하는 박테리아의 능력들은 인식하고 그들의 환경에서 섭동에 응답 할 수있다 얼마나 잘 매우 의존적이며, 이것은 차례로 자신의 신호 전달 체계에 의존한다. 경보 시스템 박테리아 인코딩의 수는 &quot;미생물 IQ&quot;라고되어 그 환경의 변화와 여러 신호를 감지 할 수있는 능력과 미세 조정의 응답 1 두의 표시가 될 수 있습니다. 두 성분의 신호 전달 시스템 (TCS)은 세균에 의해 사용되는 가장 널리 시그널링 시스템이며, 그들은 외부 신호를 감지하여 음향 응답 레귤레이터 (RR) 2 인산화를 통해 송신 히스티딘 키나아제 (HK)으로 구성된다. 의 RR은 출력 영역의 다양하므로 서로 다른 음향 모드를 가질 수 있지만, 가장 일반적인 반응은 도메인 1 DNA 결합을 통해 전사 조절이다. 감지 신호 및 VAS의 대응 기능TCSS의 t의 대부분은 알 수없는 남아 있습니다. 이러한 칩 - 칩과 같은 생체 내 방법으로 일상적 전사는 3 요소 게놈 결합 부위의 측정에 사용되지만, 활성화 조건 또는 신호가 알려져있는 경우, 그들은 단지 세균이 성분계 RR들에 대해 사용될 수있다. 종종 TCS를 활성화 환경 단서는 자신의 유전자를 대상으로보다 결정하기 어렵습니다. 시험 관내 마이크로 어레이는 기재 기반 분석을 효과적으로 빠르게 유전자 표적을 결정하고 TCSS의 기능을 예측하기 위해 사용될 수있다. 상기 분석 된 RR가 인산화 따라서 아세틸 포스페이트 4와 같은 작은 분자를 사용 도너 시험 관내에서 활성화 될 수 있다는 사실을 이용한다. 이 방법에서는, DNA 친화력 - 정제 - 칩 (그림 1)에 대한 DAP 칩 이름, 관심의 RR 유전자는 E.에서 그의 태그로 복제됩니다 대장균 및 이후 정제 태그 단백질은 양방향으로 허용된다차 게놈 DNA를 전단한다. 단백질 - 결합 DNA가 다음 친화력 정화에 충실 풍부한 입력 DNA가 증폭, 형광 함께 풀링 및 사용자 관심의 유기체 (그림 1)에 만든 기와 배열에 하이브리드 레이블. 마이크로 어레이 실험 유물로 될 수 있으며, 따라서 추가 단계 분석을 최적화하기 위해 사용된다. 하나의 단계는 (그림 2에서 워크 플로우를 참조) 전기 이동성 이동 분석 (EMSA)를 사용하여 연구에서 RR에 대한 하나의 목표를 ​​결정하려고하는 것입니다. 그런 다음, 게놈 DNA와 DAP 단계에 바인딩 다음, 단백질 - 결합 및 입력 DNA 따라서 최적의 바인딩 상태를 확인한, 긍정적 인 목표는 입력 부분에 상대적으로 단백질 - 결합 부분에 농축되어 있는지 qPCR에 의해 검사된다 RR (그림 2). 어레이 혼성화 후, 데이터는 게놈 궤적을 나타내는 높은 강도 신호의 피크를 찾기 위해 분석된다 여기서 단백질 H광고는 바인딩. 함수 얻어진 유전자 표적에 근거 RR 대해 예측 될 수있다. 대상 게놈 유전자 좌는 다음 실험적 EMSAs (그림 2)를 사용하여 유효성을 검사 결합 부위의 모티브를 예측하는 데 사용됩니다. RR의 기능 예측 및 유전자 목표는 밀접하게 유사한 결합 모티프에 대한 사람들의 게놈 (그림 2)를 스캔하여 orthologous의 RR을 인코딩 종 관련 확장 할 수있다. DAP 칩 방법은 이전에 아무도 없었다 TCS에 대한 풍부한 정보를 제공 할 수 있습니다. 상기 방법은 또한 단백질이 정제 될 수 있으며 DNA 결합 상태가 결정될 수 있으면 어떠한 전사 조절을 위해 사용하고, 게놈 서열과 관심있는 유기체에 해당 될 수있다. <img alt="그림 1" fo:content-width="5in" src="/files/ftp_upload/51715/51715fig1highres.jpg" width="500" /> 그림 1. DNA 친화력 - 정제 - 칩 (DAP-칩) 전략 7. 그 유기체에서 RR 유전자는 E.에 카르복시 말단 그의 태그로 복제됩니다 대장균 발현 균주. 정제 그의 태그가 단백질 아세틸 포스페이트 인산화에 의해 활성화하고, 쉬어 된 게놈 DNA와 혼합된다. 나머지는 니켈-NTA 수지를 사용하여 친 화성 정제를 실시하면서 결합 반응의 분취 량은, 입력 DNA로 저장된다. 입력 및 RR 바인딩 된 DNA는 증폭 된 전체 게놈 있으며, 각각를 Cy3 및 Cy5에,로 표지. 표지 된 DNA를 함께 풀링하고 유전자 표적을 결정하기 위해 분석된다 타일링 어레이에 혼성화된다. 그림 수정 및 크리에이티브 커먼즈 라이센스를 7에서 사용하는 재판. <img alt="그림 2" fo:content-width="5in" src="/files/ftp_upload/51715/51715fig2highres.jpg" width="500" /> 워크 플로의 그림 2. 요약. 모든 정제 태그 PROTEI 위해N, EMSA를 이용하여 대상을 결정함으로써 시작한다. (WGA) 농축 및 입력 DNA 증폭 게놈 DNA 다음 DNA 친화력 - 정화 (DAP) 및 전체 게놈을 결합하는 단백질을 허용합니다. 표적 유전자가 알려진 경우, 알려진 타겟 단백질 - 결합 분획에 농축되도록 qPCR에를 사용한다. 어떤 대상을 판별 할 수 있다면, DNA 라벨 및 배열 하이브리드로 직접 진행합니다. qPCR에 의한 농축이 관찰 할 수없는 경우, 결합 단백질 - gDNA를 다른 단백질의 양을 사용하여 DAP-WGA의 단계를 반복합니다. 피크를 찾아 유전자를 대상에 매핑하는 배열 분석을 사용합니다. 결합 부위 모티프를 예측하는 표적 유전자의 상류 영역을 사용한다. 실험적으로 EMSAs를 사용하여 주제를 유효성을 검사합니다. 연구에서 RR의 orthologs를 인코딩 관련 종의 게놈을 스캔 모티브를 사용하고,뿐만 아니라 그 종을 대상으로 유전자를 예측하고있다. 얻어진 유전자 타겟에 기초하여, RR과 orthologs의 생리적 기능은 예측 될 수있다. 그림 수정하고 창조적 인 C를 사용하여 다시 인쇄ommons 7에서 라이센스.

<table><tbody><tr><td>HisTrapFF column (Ni-Sepharose column)</td><td>GE Lfe Sciences, Pittsburgh, PA, USA</td><td>17-5255-01</td><td></td></tr><tr><td>Akta explorer (FPLC instrument)</td><td>GE Lfe Sciences, Pittsburgh, PA, USA</td><td></td><td></td></tr><tr><td>HiPrep 26/10 Desalting column</td><td>GE Lfe Sciences, Pittsburgh, PA, USA</td><td>17-5087-01</td><td></td></tr><tr><td>Qiaquick Gel extraction kit</td><td>Qiagen Inc, Valencia, CA, USA</td><td>28704</td><td></td></tr><tr><td>Biotin-labeled oligonucleotides</td><td>Integrated DNA Technologies</td><td>N/A</td><td></td></tr><tr><td>6% polyacrylamide-0.5x TBE precast mini DNA retardation gel</td><td>Life Technologies, Grand Island, NY, USA</td><td>EC63652BOX</td><td>Alternately, you can pour your own gel.</td></tr><tr><td>Nylon membrane</td><td>EMD Millipore, Billerica, MA, USA</td><td>INYC00010</td><td></td></tr><tr><td>Trans-Blot SD Semi-dry electrophoretic transfer cell</td><td>Biorad, Hercules, CA, USA</td><td>170-3940</td><td></td></tr><tr><td>Extra thick blot paper, 8 x 13.5 cm</td><td>Biorad, Hercules, CA, USA</td><td>170-3967</td><td></td></tr><tr><td>UV crosslinker Model XL-1000</td><td>Fisher Scientific</td><td>11-992-89</td><td></td></tr><tr><td>Nucleic Acid chemiluminescent detection kit (Pierce)</td><td>Thermo fisher Scientific, Rockford, IL, USA</td><td>89880</td><td></td></tr><tr><td>Ni-NTA agarose resin</td><td>Qiagen Inc, Valencia, CA, USA</td><td>30210</td><td></td></tr><tr><td>GenomePlex Whole genome amplification kit (Fragmentation buffer, library preparation buffer, library stabilization solution, library preparation enzyme, 10x amplification master mix, WGA polymerase)</td><td>Sigma-Aldrich, St. Louis, MO, USA</td><td>WGA2-50RXN</td><td></td></tr><tr><td>Nanodrop ND-1000</td><td>Thermo Scientific, Wilmington, DE, USA</td><td>For quantitation of DNA</td><td></td></tr><tr><td>Perfecta Sybr Green SuperMix, with ROX</td><td>Quanta biosciences</td><td>95055-500</td><td>Any Sybr Green PCR mix may be used</td></tr><tr><td>PlateMax Ultra clear heat sealing film for qPCR</td><td>Axygen</td><td></td><td></td></tr><tr><td colspan="4">[header]</td></tr><tr><td>96-well clear low profile PCR microplate</td><td>Life Technologies, Grand Island, NY, USA</td><td>PCR-96-LP-AB-C</td><td></td></tr><tr><td>Applied Biosystems StepOne Plus Real time PCR system</td><td>Life Technologies, Grand Island, NY, USA</td><td>4376600</td><td>Any real time PCR system may be used</td></tr><tr><td>Qiaquick PCR purification kit</td><td>Qiagen Inc, Valencia, CA, USA</td><td>28104</td><td>Any PCR clean up kit may be used</td></tr><tr><td>Cy3/Cy5-labeled nonamers</td><td>Trilink biotechnologies, San Diego, CA, USA</td><td>N46-0001, N46-0002</td><td></td></tr><tr><td>Klenow polymerase 50,000 U/ml, 3&#039;-5&#039; exo&lt;sup&gt;-&lt;/sup&gt;</td><td>New England Biolabs, Ipswich, MA</td><td>M0212M</td><td></td></tr><tr><td>Hybridization system</td><td>Roche-Nimblegen, Madison, WI, USA</td><td>N/A</td><td>This company no longer makes arrays or related items, so alternate sources such as Agilent or Affymetrix will need to be used.</td></tr><tr><td>Custom printed microarrays and mixers</td><td>Roche-Nimblegen, Madison, WI, USA</td><td>N/A</td><td></td></tr><tr><td>Hybridization kit (2x Hybridization buffer, Hybridization component A, Alignment oligo)</td><td>Roche-Nimblegen, Madison, WI, USA</td><td>N/A</td><td></td></tr><tr><td>Wash buffer kit (10x Wash buffer I, II, III, 1 M DTT)</td><td>Roche-Nimblegen, Madison, WI, USA</td><td>N/A</td><td></td></tr><tr><td>GenePix 4200A microarray scanner</td><td>Molecular Devices, Sunnyvale CA, USA</td><td>This model has been replaced by superior ones</td><td></td></tr><tr><td>GenePix Pro microarray software</td><td>Molecular Devices, Sunnyvale CA, USA</td><td></td><td></td></tr><tr><td>Nimblescan v.2.4, ChIP-chip analysis software</td><td>Roche-Nimblegen, Madison, WI, USA</td><td>N/A</td><td></td></tr></tbody></table>

dna-affinity-purified chip (dap-chip) method to determine gene targets for bacterial two component regulatory systems

이러한 칩 칩으로 생체 방법에서 전사 인자 글로벌 유전자 목표를 결정하는 데 사용 잘 확립 기술은 다음과 같습니다. 그러나, 그들은에 uncharacterized 활성화 조건 세균성 두 구성 요소 규제 시스템을 탐험에 사용이 제한됩니다. 독특한 신호들의 존재하에 활성화 된 때, 그러한 시스템에만 전사를 조절한다. 이들 신호는 종종 알 수 있기 때문에,이 비디오 문서에 설명 된 시험 관내 마이크로 어레이 기반의 방법은 유전자 표적 및 응답 레귤레이터 결합 부위를 결정하기 위하여 사용될 수있다. 이것은 DNA-친 화성 정제 칩 방법은 시퀀스 된 게놈 어떤 유기체 어떠한 레귤레이터 정제에 사용될 수있다. 프로토콜은 정제 태그 단백질은 사용자 정의 타일 배열에 DNA와 하이브리드의 형광 표지 다음에 단백질 바인딩 DNA를 깎인 게놈 DNA에 결합하고 친화력 정화 할 수 있도록 포함한다. 이 사양에 대한 분석을 최적화하기 위해 사용될 수있다 같이 전일C 규제도 설명되어 있습니다. 어레이 데이터 분석에 의해 생성 된 피크 후 실험적으로 검증되는 결합 부위 모티프를 예측하는 데 사용된다. 모티프 예측 더욱 밀접한 관련 종 orthologous 반응 조절제의 유전자 표적을 결정하기 위하여 사용될 수있다. 우리는 유전자의 목표를 ​​결정하고 사이트의 모티브를 결합함으로써 환경 박테리아 Desulfovibrio 심상 성 Hildenborough에서 sigma54 의존 응답 레귤레이터 DVU3023의 기능을 예측하여이 방법의 적용 가능성을 보여줍니다.

상기 방법은 Desulfovibrio 불가리스 Hildenborough 7 박테리아를 감소 모델 황산에서의 RR 해외 유전자 표적을 결정하기 위해 적용 하였다. 이 유기체가 그것을 감지에 응답 가능한 신호의 다양한 나타내는 70의 RR로 표현 TCSS의 다수가 있습니다. 생체 이러한 경보 시스템의 기능을 분석하고 자신의 신호 때문에 수행하기 어렵고 따라서 자신의 활성화 조건입니다 알 수 있습니다. 여기...

Watch this Scientific Journal Video about DNA-affinity-purified Chip DAP-chip Method to Determine Gene Targets for Bacterial Two component Regulatory Systems at JoVE.com

DNA-affinity-purified Chip DAP-chip Method to Determine Gene Targets for Bacterial Two component Regulatory Systems

이 비디오 문서는 두 개의 구성 요소 시스템 응답 레귤레이터 유전자 표적 및 결합 부위를 결정하기 위해 시험 관내 마이크로 어레이 기반의 방법을 설명한다.

세균에 대한 두 개의 구성 요소 규제 시스템을 유전자 표적을 확인하는 DNA 친화력 - 정제 칩 (DAP-칩) 방법

In vivo methods such as ChIP-chip are well-established techniques used to determine global gene targets for transcription factors. However, they are of ...

dna-affinity-purified-chip-dap-chip-method-to-determine-gene-targets

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JoVE Journal

Biology

DNA-affinity-purified Chip (DAP-chip) Method to Determine Gene Targets for Bacterial Two component Regulatory Systems

16.7K 조회수.  Lawrence Berkeley National Laboratory.  이 비디오 문서는 두 개의 구성 요소 시스템 응답 레귤레이터 유전자 표적 및 결합 부위를 결정하기 위해 시험 관내 마이크로 어레이 기반의 방법을 설명한다. 

비디오: DNA-affinity-purified Chip DAP-chip Method to Determine Gene Targets for Bacterial Two component Regulatory Systems

A Chromatin Immunoprecipitation Assay to Identify Novel NFAT2 Target Genes in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant B cell clones and represents the most common leukemia in western countries. The majority of CLL patients show an indolent course of the disease as well as an anergic phenotype of their leukemia cells, referring to a B cell receptor unresponsive to external stimulation. We have recently shown that the transcription factor NFAT2 is a crucial regulator of anergy in CLL. A major challenge in the analysis of the role of a transcription factor in different diseases is the identification of its specific target genes. This is of&#160;great significance for the elucidation of pathogenetic mechanisms and potential therapeutic interventions. Chromatin immunoprecipitation (ChIP) is a classic technique to demonstrate protein-DNA interactions and can, therefore, be used to identify direct target genes of transcription factors in mammalian cells. Here, ChIP was used to identify LCK as a direct target gene of NFAT2 in human CLL cells. DNA and associated proteins are crosslinked using formaldehyde and subsequently sheared by sonication into DNA fragments of approximately 200-500 base pairs (bp). Cross-linked DNA fragments associated with NFAT2 are then selectively immunoprecipitated from cell debris using an &#945;NFAT2 antibody. After purification, associated DNA fragments are detected via quantitative real-time PCR (qRT-PCR). DNA sequences with evident enrichment represent regions of the genome which are targeted by NFAT2 in vivo. Appropriate shearing of the DNA and the selection of the required antibody are particularly crucial for the successful application of this method. This protocol is ideal for the demonstration of direct interactions of NFAT2 with target genes. Its major limitation is the difficulty to employ ChIP in large-scale assays analyzing the target genes of multiple transcription factors in intact organisms.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the western world. NFAT transcription factors are important regulators of development and activation in numerous cell types. Here, we present a protocol for the use of chromatin immunoprecipitation (ChIP) in human CLL cells to identify novel target genes of NFAT2.

만성 림프모구 백혈병에서 소설 NFAT2 대상 유전자를 식별 하는 Chromatin Immunoprecipitation 분석 결과

Chronic lymphocytic leukemia (CLL) is characterized by the expansion of malignant B cell clones and represents the most common leukemia in western ...

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암 연구학

A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some factors act directly on virulence genes; others control pathogenesis by adjusting the expression of downstream regulators or the accumulation of signals that affect regulator activity. While regulation has been studied extensively during in vitro growth, relatively little is known about how gene expression is adjusted during infection. Such information is important when a particular gene product is a candidate for therapeutic intervention. Transcriptional approaches like quantitative, real-time RT-PCR and RNA-Seq are powerful ways to examine gene expression on a global level but suffer from many technical challenges including low abundance of bacterial RNA compared to host RNA, and sample degradation by RNases. Evaluating regulation using fluorescent reporters is relatively easy and can be multiplexed with fluorescent proteins with unique spectral properties. The method allows for single-cell, spatiotemporal analysis of gene expression in tissues that exhibit complex three-dimensional architecture and physiochemical gradients that affect bacterial regulatory networks. Such information is lost when data are averaged over the bulk population. Herein, we describe a method for quantifying gene expression in bacterial pathogens in situ. The method is based on simple tissue processing and direct observation of fluorescence from reporter proteins. We demonstrate the utility of this system by examining the expression of Staphylococcus aureus thermonuclease (nuc), whose gene product is required for immune evasion and full virulence ex vivo and in vivo. We show that nuc-gfp is strongly expressed in renal abscesses and reveal heterogeneous gene expression due in part to apparent spatial regulation of nuc promoter activity in abscesses fully engaged with the immune response. The method can be applied to any bacterium with a manipulatable genetic system and any infection model, providing valuable information for preclinical studies and drug development.

Described here is a method for analyzing bacterial gene expression in animal tissues at a cellular level. This method provides a resource for studying the phenotypic diversity occurring within a bacterial population in response to the tissue environment during an infection.

감염 된 조직에서 세균 유전자 규칙을 공부 하는 형광 기반 방법

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some ...

유전학

Stress-induced Antibiotic Susceptibility Testing on a Chip

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria survive these stressful conditions. The hypothesis behind this method is new: stress activation of biochemical pathways, which are targets of antibiotics, can accelerate antibiotic susceptibility testing. As compared to standard antibiotic susceptibility testing methods, the rate-limiting step -&#160;bacterial growth -&#160;is omitted during antibiotic application. The technical implementation of the method is in a combination of standard techniques and innovative approaches. The standard parts of the method include bacterial culture protocols, defining microfluidic channels in polydimethylsiloxane&#160;(PDMS), cell viability monitoring with fluorescence, and batch image processing for bacteria counting. Innovative parts of the method are in the use of culture media flow for mechanical stress application, use of enzymes to damage but not kill the bacteria, and use of microarray substrates for bacterial attachment. The developed platform can be used in antibiotic and nonantibiotic related drug development and testing. As compared to the standard bacterial suspension experiments, the effect of the drug can be turned on and off repeatedly over controlled time periods. Repetitive observation of the same bacterial population is possible over the course of the same experiment.

We have developed a microfluidic platform for rapid antibiotic susceptibility testing. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria can survive these stressful conditions.

칩에 스트레스 유발 항생제 감수성 테스트

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over ...

생체공학

An Assay for Quantifying Protein-RNA Binding in Bacteria

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding of an RNA binding protein (RBP) to the initiation region of the mRNA, which interferes with ribosome binding. In the presented method, we utilize this blocking phenomenon to quantify the binding affinity of RBPs to their cognate and non-cognate binding sites. To do this, we insert a test binding site in the initiation region of a reporter mRNA and induce the expression of the test RBP. In the case of RBP-RNA binding, we observed a sigmoidal repression of the reporter expression as a function of RBP concentration. In the case of no-affinity or very low affinity between binding site and RBP, no significant repression was observed. The method is carried out in live bacterial cells, and does not require expensive or sophisticated machinery. It is useful for quantifying and comparing between the binding affinities of different RBPs that are functional in bacteria to a set of designed binding sites. This method may be inappropriate for binding sites with high structural complexity. This is due to the possibility of repression of ribosomal initiation by complex mRNA structure in the absence of RBP, which would result in lower basal reporter gene expression, and thus less-observable reporter repression upon RBP binding.

In this method, we quantify the binding affinity of RNA binding proteins (RBPs) to cognate and non-cognate binding sites using a simple, live, reporter assay in bacterial cells. The assay is based on repression of a reporter gene.

박테리아에서 단백질 RNA 결합을 정량화하기위한 분석

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding ...

Discovering CsgD Regulatory Targets in Salmonella Biofilm Using Chromatin Immunoprecipitation and High-Throughput Sequencing (ChIP-seq)

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a technique that can be used to discover the regulatory targets of transcription factors, histone modifications, and other DNA-associated proteins. ChIP-seq data can also be used to find differential binding of transcription factors in different environmental conditions or cell types. Initially, ChIP was performed through hybridization on a microarray (ChIP-chip); however, ChIP-seq has become the preferred method through technological advancements, decreasing financial barriers to sequencing, and massive amounts of high-quality data output. Techniques of performing ChIP-seq with bacterial biofilms, a major source of persistent and chronic infections, are described in this protocol. ChIP-seq is performed on Salmonella enterica serovar Typhimurium biofilm and planktonic cells, targeting the master biofilm regulator, CsgD, to determine differential binding in the two cell types. Here, we demonstrate the appropriate amount of biofilm to harvest, normalizing to a planktonic control sample, homogenizing biofilm for cross-linker access, and performing routine ChIP-seq steps to obtain high quality sequencing results.

Discovering CsgD Regulatory Targets in Salmonella Biofilm Using Chromatin Immunoprecipitation and High-Throughput Sequencing ChIP-seq

Chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) is a method used to establish interactions between transcription factors and the genomic sequences they control. This protocol outlines techniques for performing ChIP-seq with bacterial biofilms, using Salmonella enterica serovar Typhimurium bacterial biofilm as an example.

크로마틴 면역침전 및 고처리량 시퀀싱(ChIP-seq)을 이용한 살모넬라 바이오필름의 CsgD 규제 목표 발견

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a technique that can be used to discover the regulatory targets of transcription ...

Gene Expression Profiling of Infecting Microbes Using a Digital Bar-coding Platform

For most mammalian pathogens, gene expression profiling studies have been limited by technical difficulties to accurately quantify pathogen gene transcripts from infected tissues. Pathogen RNA constitutes a tiny portion of the total RNA isolated from infected tissue samples. Both microarray and RNAseq technologies have difficulties in generating reliable reads for weakly expressed pathogen genes. Mutant pathogen strains with reduced in vivo proliferation pose an even bigger challenge. Here we describe an in vivo gene expression profiling protocol that is very fast, extremely sensitive and highly reproducible. We developed this protocol during our investigation of the fungal pathogen Candida albicans in a murine model of hematogenously disseminated candidiasis. Using this protocol, we have documented time courses of dynamically regulated C. albicans gene expression during kidney infection, and discovered unexpected features of gene expression responses to antifungal drug treatment in vivo.

We developed a protocol that is fast, sensitive and reproducible for pathogen gene expression profiling during an infection.

디지털 바 코딩 플랫폼을 사용에 감염 미생물의 유전자 발현 프로파일 링

For most mammalian pathogens, gene expression profiling studies have been limited by technical difficulties to accurately quantify pathogen gene ...

면역학 및 감염병학

Pulldown Assay Coupled with Co-Expression in Bacteria Cells as a Time-Efficient Tool for Testing Challenging Protein-Protein Interactions

Pulldown is an easy and widely used protein-protein interaction assay. However, it has limitations in studying protein complexes that do not assemble effectively in vitro. Such complexes may require co-translational assembly and the presence of molecular chaperones; either they form stable oligomers which cannot dissociate and re-associate in vitro or are unstable without a binding partner. To overcome these problems, it is possible to use a method based on the bacterial co-expression of differentially tagged proteins using a set of compatible vectors followed by the conventional pulldown techniques. The workflow is more time-efficient compared to traditional pulldown because it lacks the time-consuming steps of separate purification of interacting proteins and their following incubation. Another advantage is a higher reproducibility due to a significantly smaller number of steps and a shorter period of time in which proteins that exist within the in vitro environment are exposed to proteolysis and oxidation. The method was successfully applied for studying a number of protein-protein interactions when other in vitro techniques were found to be unsuitable. The method can be used for batch testing protein-protein interactions. Representative results are shown for studies of interactions between BTB domain and intrinsically disordered proteins, and of heterodimers of zinc-finger-associated domains.

Here, we describe a method for the bacterial co-expression of differentially tagged proteins using a set of compatible vectors, followed by the conventional pulldown techniques to study protein complexes that cannot assemble in vitro.

까다로운 단백질-단백질 상호작용을 테스트하기 위한 시간 효율적인 도구로서 박테리아 세포의 공동 발현과 결합된 풀다운 분석

Pulldown is an easy and widely used protein-protein interaction assay. However, it has limitations in studying protein complexes that do not assemble ...

생화학

<meta charset="utf8"></head><body>판별 RNA 및 DNA 분석을 위한 정밀 DNAzyme 나노머신

DNAzyme 10-23 - Based Nanomachines for Nucleic Acid Recognition

DNAzyme-based nanomachines (DNM) for the detection of DNA and RNA sequences (analytes) are multifunctional structures made of oligonucleotides. Their functions include tight analyte binding, highly selective analyte recognition, fluorescent signal amplification by multiple catalytic cleavages of a fluorogenic reporter substrate, and fluorogenic substrate attraction for an increase in sensor response. Functional units are attached to a common DNA scaffold for their cooperative action. The RNA-cleaving 10-23 DNMs feature&#160;improved sensitivity in comparison with non-catalytic hybridization probes. The stability of the DNM and the increased chances of substrate recognition are provided&#160;by a double-stranded DNA fragment,&#160;a tile. DNM can differentiate two analytes with a single nucleotide difference in a folded RNA and a double-stranded DNA and detect analytes at concentrations ~1000 times lower than other protein-free hybridization probes. This article presents the concept behind the diagnostic potential of DNA-nanomachine activity and overviews DNM design, assembly, and application&#160;in nucleic acid detection assays.

<meta charset="utf8"></head><body>저자 스포트라이트: DNA 나노센서의 발전 – 분자 검출의 감도 및 선택성 문제 해결

The DNAzyme-based nanomachines can be used for highly selective and sensitive detection of nucleic acids. This article describes a detailed protocol for the design of DNAzyme-based nanomachines with a 10-23 core using free software and their application in the detection of an Epstein-Barr virus fragment as an example.

DNAzyme 10-23 - 핵산 인식을 위한 기반 나노머신

DNAzyme-based nanomachines (DNM) for the detection of DNA and RNA sequences (analytes) are multifunctional structures made of oligonucleotides. Their ...

세균에 대한 두 개의 구성 요소 규제 시스템을 유전자 표적을 확인하는 DNA 친화력 - 정제 칩 (DAP-칩) 방법

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만성 림프모구 백혈병에서 소설 NFAT2 대상 유전자를 식별 하는 Chromatin Immunoprecipitation 분석 결과

감염 된 조직에서 세균 유전자 규칙을 공부 하는 형광 기반 방법

칩에 스트레스 유발 항생제 감수성 테스트

박테리아에서 단백질 RNA 결합을 정량화하기위한 분석

크로마틴 면역침전 및 고처리량 시퀀싱(ChIP-seq)을 이용한 살모넬라 바이오필름의 CsgD 규제 목표 발견

디지털 바 코딩 플랫폼을 사용에 감염 미생물의 유전자 발현 프로파일 링

까다로운 단백질-단백질 상호작용을 테스트하기 위한 시간 효율적인 도구로서 박테리아 세포의 공동 발현과 결합된 풀다운 분석

DNAzyme 10-23 - 핵산 인식을 위한 기반 나노머신

DNAzyme 10-23 - 핵산 인식을 위한 기반 나노머신

DNAzyme 10-23 - 핵산 인식을 위한 기반 나노머신