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

Universidad de Cartagena UDEC

Research

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 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 ...

연구

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 ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

변조 C 디 GMP가에서 시그널링 그건 작은 분자를 스크리닝하기위한 높은 처리량 설정 구축<em&gt; 녹농균</em

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

면역학 및 감염병학

Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the protein domains that bind to specific histone modifications. One such domain is the plant homeodomain (PHD), found in several chromatin-binding proteins. The epigenetic factor RBP2 has multiple PHD domains, however, they have different functions (Figure 4). In particular, the C-terminal PHD domain, found in a RBP2 oncogenic fusion in human leukemia, binds to trimethylated lysine 4 in histone H3 (H3K4me3)1. The transcript corresponding to the RBP2 isoform containing the C-terminal PHD accumulates during differentiation of promonocytic, lymphoma-derived, U937 cells into monocytes2. Consistent with both sets of data, genome-wide analysis showed that in differentiated U937 cells, the RBP2 protein gets localized to genomic regions highly enriched for H3K4me33. Localization of RBP2 to its targets correlates with a decrease in H3K4me3 due to RBP2 histone demethylase activity and a decrease in transcriptional activity. In contrast, two other PHDs of RBP2 are unable to bind H3K4me3. Notably, the C-terminal domain PHD of RBP2 is absent in the smaller RBP2 isoform4. It is conceivable that the small isoform of RBP2, which lacks interaction with H3K4me3, differs from the larger isoform in genomic location. The difference in genomic location of RBP2 isoforms may account for the observed diversity in RBP2 function. Specifically, RBP2 is a critical player in cellular differentiation mediated by the retinoblastoma protein (pRB). Consistent with these data, previous genome-wide analysis, without distinction between isoforms, identified two distinct groups of RBP2 target genes: 1) genes bound by RBP2 in a manner that is independent of differentiation; 2) genes bound by RBP2 in a differentiation-dependent manner.
To identify differences in localization between the isoforms we performed genome-wide location analysis by ChIP-Seq. Using antibodies that detect both RBP2 isoforms we have located all RBP2 targets. Additionally we have antibodies that only bind large, and not small RBP2 isoform (Figure 4). After identifying the large isoform targets, one can then subtract them from all RBP2 targets to reveal the targets of small isoform. These data show the contribution of chromatin-interacting domain in protein recruitment to its binding sites in the genome.

Here we are presenting a chromatin immunoprecipitation (ChIP) procedure for genome-wide location analysis of protein isoforms that differ in a histone-binding domain. We are applying it to ChIP-Seq analysis to identify the targets of the KDM5A/JARID1A/RBP2 histone demethylase.

이소형 특정 유전자 목표를 식별 칩을 사용하여 게놈 차원의 분석

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the ...

생물학

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Phenotypes are determined by a complex series of physical (e.g. protein-protein) and functional (e.g. gene-gene or genetic) interactions (GI)1. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7, but GI information remains sparse for prokaryotes8, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10.
Here, we present the key steps required to perform quantitative E. coli Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format. Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g. the 'Keio' collection11) and essential gene hypomorphic mutations (i.e. alleles conferring reduced protein expression, stability, or activity9, 12, 13) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e. slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2 as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9.

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

에 박테리아 기능 네트워크 및 경로를 매핑<em&gt; 대장균</em&gt; 합성 유전자 배열을 사용하여

Phenotypes  are determined by a complex series of physical (e.g. protein-protein) and  functional (e.g. gene-gene or genetic) interactions (GI)1. While ...

A Fast and Reliable Pipeline for Bacterial Transcriptome Analysis Case study: Serine-dependent Gene Regulation in Streptococcus pneumoniae

Gene expression and its regulation are very important to understand the behavior of cells under different conditions. Various techniques are used nowadays to study gene expression, but most are limited in terms of providing an overall picture of the expression of the whole transcriptome. DNA microarrays offer a fast and economic research technology, which gives a full overview of global gene expression and have a vast number of applications including identification of novel genes and transcription factor binding sites, characterization of transcriptional activity of the cells and also help in analyzing thousands of genes (in a single experiment). In the present study, the conditions for bacterial transcriptome analysis from cell harvest to DNA microarray analysis have been optimized. Taking into account the time, costs and accuracy of the experiments, this technology platform proves to be very useful and universally applicabale for studying bacterial transcriptomes. Here, we perform DNA microarray analysis with Streptococcus pneumoniae as a case-study by comparing the transcriptional responses of S. pneumoniae grown in the presence of varying L-serine concentrations in the medium. Total RNA was isolated by using a Macaloid method using an RNA isolation kit and the quality of RNA was checked by using an RNA quality check kit. cDNA was prepared using reverse transcriptase and the cDNA samples were labelled using one of two amine-reactive fluorescent dyes. Homemade DNA microarray slides were used for hybridization of the labelled cDNA samples and microarray data were analyzed by using a cDNA microarray data pre-processing framework (Microprep). Finally, Cyber-T was used to analyze the data generated using Microprep for the identification of statistically significant differentially expressed genes. Furthermore, in-house built software packages (PePPER, FIVA, DISCLOSE, PROSECUTOR, Genome2D) were used to analyze data.

A Fast and Reliable Pipeline for Bacterial Transcriptome Analysis Case study: Serine-dependent Gene Regulation in Streptococcus pneumoniae

This manuscript describes the use of state-of-the-art technology provided by DNA-microarrays. Microarrays provide an overview of the transcriptomic changes in bacteria incurred under a specific condition. Moreover, we highlight the ease by which large amounts of data can be analyzed by using convenient in-house developed software packages.

세균의 사체 분석 사례 연구에 대한 빠르고 안정적​​인 파이프 라인 : 세린에 의존하는 유전자 조절에<em&gt; 폐렴 구균</em

Gene expression and its regulation are very important to understand the behavior of cells under different conditions. Various techniques are used nowadays ...

Functional Complementation Analysis (FCA): A Laboratory Exercise Designed and Implemented to Supplement the Teaching of Biochemical Pathways

Functional complementation assay (FCA) is an in vivo assay that is widely used to elucidate the function/role of genes/enzymes. This technique is very common in biochemistry, genetics and many other disciplines. A comprehensive overview of the technique to supplement the teaching of biochemical pathways pertaining to amino acids, peptidoglycan and the bacterial stringent response is reported in this manuscript. Two cDNAs from the model plant organism Arabidopsis thaliana that are involved in the metabolism of lysine (L,L-diaminopimelate aminotransferase (dapL) and tyrosine aminotransferase (tyrB) involved in the metabolism of tyrosine and phenylalanine are highlighted. In addition, the bacterial peptidoglycan anabolic pathway is highlighted through the analysis of the UDP-N-acetylmuramoyl-L-alanyl-D-glutamate-meso-2,6-diaminopimelate ligase (murE) gene from the bacterium Verrucomicrobium spinosum involved in the cross-linking of peptidoglycan. The bacterial stringent response is also reported through the analysis of the rsh (relA/spoT homolog) bifunctional gene responsible for a hyper-mucoid phenotype in the bacterium Novosphingobium sp. Four examples of FCA are presented. The video will focus on three of them, namely lysine, peptidoglycan and the stringent response.

Functional Complementation Analysis FCA: A Laboratory Exercise Designed and Implemented to Supplement the Teaching of Biochemical Pathways

The validation of enzymatic activities involved in biochemical pathways can be elucidated using functional complementation analysis (FCA). Described in this manuscript is the FCA assay demonstrating the enzymatic activity of enzymes involved in the metabolism of amino acids, bacterial stringent response and bacterial peptidoglycan biosynthesis.

기능 보완 분석 (FCA) : 설계 및 생화학 적 경로의 교육을 보완하기 위해 구현 된 실험실 운동

Functional complementation assay (FCA) is an in vivo assay that is widely used to elucidate the function/role of genes/enzymes. This technique is very ...

Using Microtiter Dish Radiolabeling for Multiple In Vivo Measurements Of Escherichia coli (p)ppGpp Followed by Thin Layer Chromatography

The (p)ppGpp nucleotide functions as a global regulator in bacteria in response to a variety of physical and nutritional stress. It has a rapid onset, in seconds, which leads to accumulation of levels that approach or exceed those of GTP pools. Stress reversal occasions a rapid disappearance of (p)ppGpp, often with a half-life of less than a minute. The presence of (p)ppGpp results in alterations of cellular gene expression and metabolism that counter the damaging effects of stress. Gram-negative and Gram-positive bacteria have different response mechanisms, but both depend on (p)ppGpp concentration. In any event, there is a need to simultaneously monitor many radiolabeled bacterial cultures at time intervals that may vary from 10 seconds to hours during critical stress transition periods. This protocol addresses this technical challenge. The method takes advantage of temperature- and shaker-controlled microtiter dish incubators that allow parallel monitoring of growth (absorbance) and rapid sampling of uniformly phosphate-radiolabeled cultures to resolve and quantitate nucleotide pools by thin-layer chromatography on PEI-cellulose. Small amounts of sample are needed for multiple technical and biological replicates of analyses. Complex growth transitions, such as diauxic growth and rapid (p)ppGpp turnover rates can be quantitatively assessed by this method.

Using Microtiter Dish Radiolabeling for Multiple In Vivo Measurements Of Escherichia coli pppGpp Followed by Thin Layer Chromatography

The growth of radiolabeled bacterial cultures in microtiter dishes facilitates high throughput sampling that allows multiple technical and biological replicate assays of nucleotide pool abundance, including that of (p)ppGpp. The effects of growth transitions provoked by sources of physiological stress as well as recovery from stress can be monitored.

대장균 (p)ppGpp의 여러 생체 내 측정을위한 마이크로 티터 접시 방사성 라벨링 사용 및 얇은 층 크로마토그래피

The (p)ppGpp nucleotide functions as a global regulator in bacteria in response to a variety of physical and nutritional stress. It has a rapid onset, in ...

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

요약

더 많은 비디오 탐색

이 비디오의 챕터

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

변조 C 디 GMP가에서 시그널링 그건 작은 분자를 스크리닝하기위한 높은 처리량 설정 구축

이소형 특정 유전자 목표를 식별 칩을 사용하여 게놈 차원의 분석

에 박테리아 기능 네트워크 및 경로를 매핑

세균의 사체 분석 사례 연구에 대한 빠르고 안정적인 파이프 라인 : 세린에 의존하는 유전자 조절에

기능 보완 분석 (FCA) : 설계 및 생화학 적 경로의 교육을 보완하기 위해 구현 된 실험실 운동

대장균 (p)ppGpp의 여러 생체 내 측정을위한 마이크로 티터 접시 방사성 라벨링 사용 및 얇은 층 크로마토그래피

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

요약

더 많은 비디오 탐색

이 비디오의 챕터

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

변조 C 디 GMP가에서 시그널링 그건 작은 분자를 스크리닝하기위한 높은 처리량 설정 구축

이소형 특정 유전자 목표를 식별 칩을 사용하여 게놈 차원의 분석

에 박테리아 기능 네트워크 및 경로를 매핑

세균의 사체 분석 사례 연구에 대한 빠르고 안정적​​인 파이프 라인 : 세린에 의존하는 유전자 조절에

기능 보완 분석 (FCA) : 설계 및 생화학 적 경로의 교육을 보완하기 위해 구현 된 실험실 운동

대장균 (p)ppGpp의 여러 생체 내 측정을위한 마이크로 티터 접시 방사성 라벨링 사용 및 얇은 층 크로마토그래피

세균의 사체 분석 사례 연구에 대한 빠르고 안정적인 파이프 라인 : 세린에 의존하는 유전자 조절에