Name: 'bioluminescent' reporter phage for the detection of category a bacterial pathogens
Uploaded: 2011-07-08T12:00:00.000+00:00
Duration: 11 min 31 s
Description: Watch this Scientific Journal Video about 'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens at JoVE.com

이 연구는 국립 보건원의 중소 기업 혁신 연구 프로그램 (NIAID, 1R43AI082698 - 01)과 식량 농업의 국립 연구소 USDA (NIFA, 2009-33610-20028)에 의해 지원되었다.

1. Y. pestis 플레이트 접종 <ol><li> 리크 Y. pestis A1122 (BeiResources # NR15) Luria - Bertani에 재고 문화 (LB) 한천 (밀러). 무균 기술을 사용하여 모든 Y.을 수행 클래스 II 타입 biosafety 캐비닛. Y. pestis의 조작에 pestis A1122은 BioSafety 수준 (BSL) 2 제외를 선택 에이전트 부담입니다. 그것은 모두 75킬로바이트 쌍은 낮은 칼슘 플라스미드 응답 (LCR) 독성 및 독성에 필요한 PGM의 로커스 부족하다. Y. 그로 28 pestis ° C 공기 배양기를 제어 정적 온도 48 H하십시오. 와이의 성장률 pestis는 H 9 1.25의 세대 시간 느립니다. </li></ol> 2. Y. pestis 액체 미디어 접종 <ol><li> 단일 Y.을 전송 멸균 금속 접종 루프를 사용하여 LB 제품 국물 2 ML을 포함하는 멸균 17 X 100mm 문화 튜브에 pestis 콜로니. 균일한 크기의는 식민지를 선택하고, 다른 식민지는 지표이며, 명확하게 접시에있는 식민지의 나머지 부분에서 분리됩니다. 소용돌이 간략 튜브 28에서 문화를 성장 ° C 떨고 인큐베이터 (225 RPM)에 약 20 H하십시오. </li></ol> 3. Y. pestis의 파생물, 기자 파지 추가하고, 발광 생물 검출 <ol><li> 밤새 Y.을 희석 pestis 문화 50 ML 팔콘 튜브에 신선한 LB의 국물 (예 : 매체의 9.5 ML로 세포 500 μL)로 1시 20분과 ° C (225 RPM) 잡고 28 성장. 0.2 600 NM (600)에서 광학 밀도 (약 5 H)에 도달할 때까지 성장합니다. 적극적으로 성장 세포는 발광 생물 응답 호스트 박테리아의 생존 및 피트니스에 상관이다 transduce로 파지의 능력부터 감지 바람직합니다. 그럼에도 불구하고, 탐지 시스템은 성장주기 7 모든 단계에서 수확 전지와 호환되도록 표시되었습니다. </li><li> 2 % N - 지구장의 솔루션을 준비하여 발광 생물 반응에 필요한 기판을 생성합니다. LB의 국물의 9.8 ML과 N - 지구장의 200 μL를 섞습니다. 5 S. 위해 적극적으로 소용돌이 프라임 2퍼센트 N - 지구장의 솔루션 2 ML과 microplate luminometer 주입기. luminometer 잘하고 다음 각 microplate에 지구장의 솔루션을 67 μL를 autoinject 즉시 10 S.에 대한 샘플을 읽을 사전 설정된 </li><li> 3 문화 튜브에 LB의 국물 1 ML aliquots 하는걸. 각각의 튜브에 기자 파지 주식 솔루션 (5 X 10 9 플라크 형성 단위의 주식 [PFU] / ML) 20 μL를 추가, 파지 및 미디어 혼자 샘플은 부정적인 제어 역할을합니다. 와이의 부재에 pestis 세포는 기자의 파지의 추가는 발광 생물 반응을 이끌어해서는 안됩니다. </li><li> Y. 1 ML aliquots 하는걸 6 문화 튜브에 pestis 문화 (단계 3.1에서). 문화 (테스트 문화)의 3 기자 파지 주식 20 μL를 추가합니다. 나머지 세 문화는 &#39;셀 혼자&#39;대조군 (배경 autobioluminescence)로 제공하고 있습니다. 간략 vortexing으로 문화를 믹스하고, 각 샘플에서 수확 200 μL (시간 0 읽기)과 흰색 96 - 웰 플레이트 microtiter로 분배. 28에 남아있는 문화를 품어 ° C를 (225 RPM) 잡고. </li><li> microplate의 luminometer를 사용하여 bioluminescence (상대적인 빛의 단위, RLU)에 대한 시간 0 명 샘플을 측정합니다. </li><li> 10 일 이후 각 샘플의 수확 200 μL, 20, 30, 40, 50, 그리고 60 분 후 기자 파지 추가하고, bioluminescence에 대한 샘플을 측정합니다. 경우 Y. pestis 세포가 존재, 기자의 파지는 특히 세포에 바인딩의 DNA를 삽입하고, luxAB 기자 유전자 (그림 1)을 녹음하고 번역하는 호스트에게 transcriptional과 translational 기계를 사용합니다. </li><li> 신호의 강도와 신호 응답 시간은 현재 세포의 수에 비례합니다. 셀 (10 5 -10 8 CFU / ML), 컨트롤에 비해 RLU에 상당한 증가의 높은 농도에서 20 분 이내에 분명해야합니다. 낮은 세포 농도에서, (10 2 -10 4 CFU / ML), RLU 크게 증가 40-60 분 이내에 분명해야합니다. </li><li> 또는, Y. 필요없이 탐지 과정을 진행할 수 있도록 갓 어른이 접시에서 pestis의 파생물, 식민지 직접 기자의 파지를 숨겨주 LB의 국물 200 μL와 1.5 ML eppendorf 튜브에 (vortexing으로) 혼합 수 있습니다. microtiter 접시에 잘으로 셀 / 파지 혼합 분배. 28 ° C에서 microtiter 접시를 부화하고 60 분 (단일 시점 전용) 이후 bioluminescence에 대한 샘플을 참조하십시오. </li></ol> 4. 대표 결과 : 기자 파지의 대표적인 시간 코스 실험 Y.의 검색을 중재 pestis는 그림 2에 그려져 있습니다. 1의 부정적인 컨트롤) 파지 혼자 (NO 세포), 또는 혼자서 2) 전지 (NO 파지) 60 분 병원 전반에 걸쳐 약 20 RLU의 bioluminescence의 기본 수준을 제공합니다ubation (기준 레벨 luminometer의 특정됩니다.) 대조적으로, 테스트 샘플 (기자의 파지 및 세포)에 대한 bioluminescence의 증가 파지 추가 후 15 분 분명하다. 신호 강도는 60 분 이상 꾸준히 증가한다. 연장 기간 (80 분)에 대한 Incubations는 호스트 세포의 중재 용해를 파지에 의한 피크 신호​​로부터 거부하는 신호가 발생합니다. 비슷한 결과는 37 배양 온도를 얻을 수있다 ° C에도 Y.을위한 최적 온도 pestis 성장 28 ° C 9. <img src="/files/ftp_upload/2740/2740fig2.jpg" alt="그림 2"/> 그림 2. 와이의 파지 - 중재 발광 생물 검출 pestis. 시간이 0, 기자의 파지와 세포가 28 ° C에서 incubated, 혼합되었고, bioluminescence가 (RLU) 시간이 지남에 모니터됨. RLU 크게 증가 (* 학생 T - 테스트, P &lt;0.05) 15 분 이내에 분명하다.

<ol>
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관심 없음 충돌 선언하지 않습니다.

이 방법은 빠르게 Y.을 감지하기 위해 기자 파지의 능력을 보여줍니다 기자 파지 이후 pestis는 교양 Y까지 발광 생물 신호 응답을 transduce 수 파지 추가 후 20 분 이내에 pestis 세포. 기자 파지도 직접 Y. 검출이 가능합니다 절연 이후 재배 7 전제 조건없이 임상 매트릭스에 pestis. 일반적으로 완료 48 H를 필요로하는 표준 파지의 용해 assays에 비해, 이것은 크게 검색 시간을 감소시킵니다. 이전 연구는 야생 형 ΦA1122의 파지는 거의 모든 천연 Y.을 lyse 수있는 증명 pestis는 격리하고, Y.에 대해 &#39;구체적인&#39;입니다 pestis 6,10,11 있지만, 일부 Y. pseudotuberculosis의 변종은 20 ° C 이상의 온도에서 6,10,12 성장하면 ΦA1122 감염될 수 표시되었습니다. 온도에 민감한 차동 자화율에 대한 이유는 알 수 있지만, 세포 표면 레이어 / 조성에서 온도 변화에 의존 아마도 때문에. 따라서 기자 파지 탐지 시스템의 잠재적인 주의해야 할 점은 밀접하게 관련 종 Y.에서 종자와 거짓 - 긍정적인 반응의 가능성 pseudotuberculosis. 제한 온도 (20 ° C)에서 기자 파지 분석을 수행하면 Y.을 포함할 수 있습니다 샘플에 잘못된 긍정적인 신호를 방지할 수 있습니다 pseudotuberculosis. 특정 온도에서 성장 고립된 문화를 사용하는 경우 따라서 특이성이 엄격하게 제어할 수 있습니다. 연 요약, 빠른 감지 및 진단에 pestis는 전염병 이후 긍정적인 예후 (豫后), 특히 호흡기 전염병에 대한 필수적인 치료가 증상 발병 후 첫 24 H 내에 실시하지 않은 경우 거의 항상 치명적이다. 이 방법은 교양 격리의 확인 식별 또는 임상 관련 매트릭스 내에서 이러한 요구를 충족시킬 잠재력이있다.

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th></tr><tr><td> Difco의 LB 한천, 밀러 </td><td> VWR </td><td> 90003-346 </td></tr><tr><td> Difco LB 국물, 밀러 </td><td> VWR </td><td> 90003-350 </td></tr><tr><td> 17 X 100mm 문화 튜브 </td><td> 미국 과학 </td><td> 1485-0810 </td></tr><tr><td> N - 지구장의 </td><td> 시그마 </td><td> D7384 </td></tr><tr><td> 베리타스 microplate의 luminometer </td><td> 터너 Biosystems </td><td> 9100-001 </td></tr><tr><td> Microlite microtiter 96 - 웰 플레이트 </td><td> VWR </td><td> 62402-984 </td></tr></tbody></table>

'bioluminescent' reporter phage for the detection of category a bacterial pathogens

Yersinia pestis의 및 바실러스 anthracis는 각각 1, 역병과 탄저병의 원인이되는 대리인하는 세균성 병원균 카테고리입니다. 두 질환 &#39;의 자연 발생 지금은 비교적 드문이지만, 분류된 생물학 무기 이러한 병원균을 사용하는 테러 집단의 가능성은 현실입니다. 질병의 고유한 communicability, 신속한 임상 과정, 높은 사망률 때문에 그것은 확산이 빠르게 감지하는 것이 중요합니다. 따라서 빠른 감지 및 진단을 제공하는 방법은 공중 보건 조치 및 위기 관리의 활성화의 즉각적인 이행을 보장하기 위해 필수적입니다. 재조합 기자 파지는 Y.의 검색에 대한 신속하고 구체적인 접근 방식을 제공할 수 있습니다 pestis와 B. anthracis가. 질병 통제 예방 센터 (CDC)는 현재 이러한 세균 병원균 2-4 확인 식별에 대한 고전적인 파지의 용해 assays를 사용합니다. 이러한 assays는 자연스럽게 자신의 세균 호스트에 대한 구체적이고 lytic 아르 파지를 발생 활용합니다. 특정 파지의 존재에 재배 세균의 하룻밤 성장 후, plaques (세균 용해)의 형성은 박테리아 타겟의 긍정적인 식별을 제공합니다. 이러한 assays는 강력한 있지만, 그들은 세 가지 결점으로 고통 : 1) 그들은 실험실을 기반으로, 2) 그들이 의심되는 시료에서 세균 분리 및 배양을 필요로하고, 3) 그들은 완료하는 데 2​​4-36 H 가져가라. 이러한 문제를 해결하기 위해 재조합 &quot;빛을 태그&quot;기자 파지는 유전자 Y.의 게놈에 장염 harveyi luxAB의 유전자를 통합하여 설계되었습니다 pestis와 B. anthracis 특정 파지 5-8. 그 결과 luxAB 기자의 파지는 빠르게하여 특정 목표를 감지할 수 있었다 (분 이내) 및 sensitively받는 세포에 발광 생물 표현형를 부여. 중요한 것은 감지 재배받는 세포 또는 모의에 감염된 임상 표본 7 중 얻은 것입니다. 데모를 위해, 우리가 알려진 Y.의 파지 - 중재 검출하는 방법을 설명 pestis는 CDC 전염병 진단 파지 ΦA1122 6,7 (그림 1)에서 만들어진 luxAB 기자 파지를 사용하여 분리. 사소한 수정 (성장 온도와 미디어 예 변화)와 유사한 방법, B.의 검색에 사용할 수 있습니다 anthracis은 B.를 사용하여 분리 anthracis 기자의 파지의 Wβ : luxAB 8. 방법은 재배 Y까지 biolumescent 표현형의 파지 - 중재 전달을 설명합니다 이후 microplate의 luminometer를 사용하여 측정 pestis 세포. 전통적인 파지의 용해 assays를 통해이 방법의 주요 장점은 사용, 빠른 결과를, 그리고 96 - 웰 플레이트 microtiter 형식으로 동시에 여러 샘플을 테스트하는 기능의 용이성이다. <img src="/files/ftp_upload/2740/2740fig1.jpg" alt="그림 1"/> 그림 1. 검색 설계도. 파지를 예제와 함께 혼합되어, 파지는 세포, luxAB가 표현되며, 세포를 감염 bioluminesces. 샘플 처리가 필요하지 않습니다, 파지와 세포가 혼합되고 이후 신호를 측정.

Watch this Scientific Journal Video about 'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens at JoVE.com

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

우선 세균성 병원균의 식별을위한 간단한 방법은 유전자 조작 기자 파지를 사용하는 것입니다. 자신의 특정 호스트 수종와 관련된 이러한 기자의 파지는 빠르게 호스트 세포에 발광 생물 신호 응답을 transducing 수 있습니다. 여기에, 우리는의 검출에 대한 기자의 파지의 사용을 설명<em&gt; Yersinia pestis</em&gt;.

카테고리의 감지 세균성 병원균에 대한 &#39;발광 생물&#39;리포터 파지

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

bioluminescent-reporter-phage-for-detection-category-bacterial

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

13.4K 조회수.  Guild Associates, Inc.. 우선 세균성 병원균의 식별을위한 간단한 방법은 유전자 조작 기자 파지를 사용하는 것입니다. 자신의 특정 호스트 수종와 관련된 이러한 기자의 파지는 빠르게 호스트 세포에 발광 생물 신호 응답을 transducing 수 있습니다. 여기에, 우리는의 검출에 대한 기자의 파지의 사용을 설명 Yersinia pestis.

비디오: 'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

마우스의 이미지 세균 감염에 루시페라제를 사용하여

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

연구

면역학 및 감염병학

In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

There is a considerable number of bacterial species capable of emitting light. All of them share the same gene cluster, namely the lux operon. Despite this similarity, these bacteria show extreme variations in characteristics like growth behavior, intensity of light emission or regulation of bioluminescence. The method presented here is a newly developed assay that combines recording of cell growth and bioluminescent light emission intensity over time utilizing a plate reader. The resulting growth and light emission characteristics can be linked to important features of the respective bacterial strain, such as quorum sensing regulation. The cultivation of a range of bioluminescent bacteria requires a specific medium (e.g., artificial sea water medium) and defined temperatures. The easy to handle, non-bioluminescent standard-research bacterium Escherichia coli (E. coli), on the other hand, can be cultivated inexpensively in high quantities in laboratory scale. Exploiting E. coli by introducing a plasmid containing the whole lux operon can simplify experimental conditions and additionally opens up many possibilities for future applications. The expression of all lux genes utilizing an E. coli expression strain was achieved by construction of an expression plasmid via Gibson cloning and insertion of four fragments containing seven lux genes and three rib genes of the lux-rib operon into a pET28a vector. E. coli based lux gene expression can be induced and controlled via Isopropyl-&#946;-D-thiogalactopyranosid (IPTG) addition resulting in bioluminescent E. coli cells. The advantages of this system are to avoid quorum sensing regulation restrictions and complex medium compositions along with non-standard growth conditions, such as defined temperatures. This system enables analysis of lux genes and their interplay, by the exclusion of the respective gene from the lux operon, or even addition of novel genes, exchanging the luxAB genes from one bacterial strain by another, or analyzing protein complexes, such as luxCDE.

In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

Bioluminescent bacteria regulate light production through a variety of mechanisms, such as quorum sensing. This novel method allows the in situ investigation of bioluminescence and the correlation of light emission to cell density. An artificial bioluminescent Escherichia coli system allows the characterization of lux operons, Lux proteins, and their interplay.

제자리에서 측정 및 셀 밀도의 상관 관계 및 발광 박테리아의 발광

There is a considerable number of bacterial species capable of emitting light. All of them share the same gene cluster, namely the lux operon. Despite ...

생화학

Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) antibody conjugated latex beads have been utilized to create a biosensor designed for discrimination of methicillin resistant (MRSA) and sensitive (MSSA) S. aureus species 1,2. The lytic phages have been converted into phage spheroids by contact with water-chloroform interface. Phage spheroid monolayers have been moved onto a biosensor surface by Langmuir-Blodgett (LB) technique 3. The created biosensors have been examined by a quartz crystal microbalance with dissipation tracking (QCM-D) to evaluate bacteria-phage interactions. Bacteria-spheroid interactions led to reduced resonance frequency and a rise in dissipation energy for both MRSA and MSSA strains. After the bacterial binding, these sensors have been further exposed to the penicillin-binding protein antibody latex beads. Sensors analyzed with MRSA responded to PBP 2a antibody beads; although sensors inspected with MSSA gave no response. This experimental distinction determines an unambiguous discrimination between methicillin resistant and sensitive S. aureus strains. Equally bound and unbound bacteriophages suppress bacterial growth on surfaces and in water suspensions. Once lytic phages are changed into spheroids, they retain their strong lytic activity and show high bacterial capture capability. The phage and phage spheroids can be utilized for testing and sterilization of antibiotic resistant microorganisms. Other applications may include use in bacteriophage therapy and antimicrobial surfaces.

Lytic phage biosensors and antibody beads are able to discriminate between methicillin resistant (MRSA) and sensitive staphylococcus bacteria. The phages were immobilized by a Langmuir-Blodgett method onto a surface of a quartz crystal microbalance sensor and worked as broad range staphylococcus probes. Antibody beads recognize MRSA.

항생제 내성 황색 포도상 구균 박테리아의 검출을위한 바이오 센서

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) ...

생체공학

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

Detection of Bacteria Using Fluorogenic DNAzymes

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses each and every year. Prevention of such outbreaks and minimization of the impact of an ongoing epidemic place an ever-increasing demand for analytical methods that can accurately identify culprit pathogens at the earliest stage. Although there is a large array of effective methods for pathogen detection, none of them can satisfy all the following five premier requirements embodied for an ideal detection method: high specificity (detecting only the bacterium of interest), high sensitivity (capable of detecting as low as a single live bacterial cell), short time-to-results (minutes to hours), great operational simplicity (no need for lengthy sampling procedures and the use of specialized equipment), and cost effectiveness. For example, classical microbiological methods are highly specific but require a long time (days to weeks) to acquire a definitive result.1 PCR- and antibody-based techniques offer shorter waiting times (hours to days), but they require the use of expensive reagents and/or sophisticated equipment.2-4 Consequently, there is still a great demand for scientific research towards developing innovative bacterial detection methods that offer improved characteristics in one or more of the aforementioned requirements. Our laboratory is interested in examining the potential of DNAzymes as a novel class of molecular probes for biosensing applications including bacterial detection.5
DNAzymes (also known as deoxyribozymes or DNA enzymes) are man-made single-stranded DNA molecules with the capability of catalyzing chemical reactions.6-8 These molecules can be isolated from a vast random-sequence DNA pool (which contains as many as 1016 individual sequences) by a process known as "in vitro selection" or "SELEX" (systematic evolution of ligands by exponential enrichment).9-16 These special DNA molecules have been widely examined in recent years as molecular tools for biosensing applications.6-8
Our laboratory has established in vitro selection procedures for isolating RNA-cleaving fluorescent DNAzymes (RFDs; Fig. 1) and investigated the use of RFDs as analytical tools.17-29 RFDs catalyze the cleavage of a DNA-RNA chimeric substrate at a single ribonucleotide junction (R) that is flanked by a fluorophore (F) and a quencher (Q). The close proximity of F and Q renders the uncleaved substrate minimal fluorescence. However, the cleavage event leads to the separation of F and Q, which is accompanied by significant increase of fluorescence intensity.
More recently, we developed a method of isolating RFDs for bacterial detection.5 These special RFDs were isolated to "light up" in the presence of the crude extracellular mixture (CEM) left behind by a specific type of bacteria in their environment or in the media they are cultured (Fig. 1). The use of crude mixture circumvents the tedious process of purifying and identifying a suitable target from the microbe of interest for biosensor development (which could take months or years to complete). The use of extracellular targets means the assaying procedure is simple because there is no need for steps to obtain intracellular targets.
Using the above approach, we derived an RFD that cleaves its substrate (FS1; Fig. 2A) only in the presence of the CEM produced by E. coli (CEM-EC).5 This E. coli-sensing RFD, named RFD-EC1 (Fig. 2A), was found to be strictly responsive to CEM-EC but nonresponsive to CEMs from a host of other bacteria (Fig. 3).
Here we present the key experimental procedures for setting up E. coli detection assays using RFD-EC1 and representative results.

We have recently reported a novel approach for generating fluorogenic DNAzyme probes that can be applied to set up a simple, "mix-and-read" fluorescent assay for bacterial detection. These special DNA probes catalyze the cleavage of a chromophore-modified DNA-RNA chimeric substrate in the presence of crude extracellular mixture (CEM) produced by a specific bacterium, thereby translating bacterial detection into fluorescence signal generation. In this report we will describe key experimental procedures where a specific DNAzyme probe denoted "RFD-EC1" is employed for the detection of the model bacterium, Escherichia coli (E. coli).

형광 DNAzymes을 사용하여 박테리아의 탐지

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses ...

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

발광 생물 세균 이미징<em&gt; 생체 내</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

A Step Beyond BRET: Fluorescence by Unbound Excitation from Luminescence (FUEL)

Fluorescence by Unbound Excitation from Luminescence (FUEL) is a radiative excitation-emission process that produces increased signal and contrast enhancement in vitro and in vivo. FUEL shares many of the same underlying principles as Bioluminescence Resonance Energy Transfer (BRET), yet greatly differs in the acceptable working distances between the luminescent source and the fluorescent entity. While BRET is effectively limited to a maximum of 2 times the F&ouml;rster radius, commonly less than 14 nm, FUEL can occur at distances up to &micro;m or even cm in the absence of an optical absorber. Here we expand upon the foundation and applicability of FUEL by reviewing the relevant principles behind the phenomenon and demonstrate its compatibility with a wide variety of fluorophores and fluorescent nanoparticles. Further, the utility of antibody-targeted FUEL is explored. The examples shown here provide evidence that FUEL can be utilized for applications where BRET is not possible, filling the spatial void that exists between BRET and traditional whole animal imaging.

A Step Beyond BRET: Fluorescence by Unbound Excitation from Luminescence FUEL

Expanding the foundation and applicability of Fluorescence by Unbound Excitation from Luminescence (FUEL) by surveying the relevant principles and demonstrating its compatibility with a multitude of fluorophores and antibody-targeted conditions.

브레 너머 단계 : 언 바운드 흥분에 의한 형광 발광에서 (연료)

Fluorescence by Unbound Excitation from Luminescence (FUEL) is a radiative excitation-emission process that produces increased signal and contrast ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome (HUS), colonize to the intestinal tract of humans. To study the detailed mechanism of EHEC colonization in vivo, it is essential to have animal models to monitor and quantify EHEC colonization. We demonstrate here a mouse-EHEC colonization model by transforming the bioluminescent expressing plasmid to EHEC to monitor and quantify EHEC colonization in living hosts. Animals inoculated with bioluminescence-labeled EHEC show intense bioluminescent signals in mice by detection with a non-invasive in vivo imaging system. After 1 and 2 days post infection, bioluminescent signals could still be detected in infected animals, which suggests that EHEC colonize in hosts for at least 2 days. We also demonstrate that these bioluminescent EHEC locate to mouse intestine, specifically in the cecum and colon, from ex vivo images. This mouse-EHEC colonization model may serve as a tool to advance the current knowledge of the EHEC colonization mechanism.

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

A detailed protocol of a mouse model for enterohemorrhagic E. coli (EHEC) colonization by using bioluminescence-labeled bacteria is presented. The detection of these bioluminescent bacteria by a non-invasive in vivo imaging system in live animals can advance our current understanding of EHEC colonization.

생물 발광 시스템 비-침략 적 비보에 의해 Murine 호스트에서 Enterohemorrhagic 대장균 식민지의 검출

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...

Using Coculture to Detect Chemically Mediated Interspecies Interactions

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by secreting metabolites. These metabolites have the potential to modulate the physiology and differentiation of their microbial neighbors and are likely important factors in the establishment and maintenance of complex microbial communities. We have developed a fluorescence-based coculture screen to identify such chemically mediated microbial interactions. The screen involves combining a fluorescent transcriptional reporter strain with environmental microbes on solid media and allowing the colonies to grow in coculture. The fluorescent transcriptional reporter is designed so that the chosen bacterial strain fluoresces when it is expressing a particular phenotype of interest (i.e. biofilm formation, sporulation, virulence factor production, etc.) Screening is performed under growth conditions where this phenotype is not expressed (and therefore the reporter strain is typically nonfluorescent). When an environmental microbe secretes a metabolite that activates this phenotype, it diffuses through the agar and activates the fluorescent reporter construct. This allows the inducing-metabolite-producing microbe to be detected: they are the nonfluorescent colonies most proximal to the fluorescent colonies. Thus, this screen allows the identification of environmental microbes that produce diffusible metabolites that activate a particular physiological response in a reporter strain. This publication discusses how to: a) select appropriate coculture screening conditions, b) prepare the reporter and environmental microbes for screening, c) perform the coculture screen, d) isolate putative inducing organisms, and e) confirm their activity in a secondary screen. We developed this method to screen for soil organisms that activate biofilm matrix-production in Bacillus subtilis; however, we also discuss considerations for applying this approach to other genetically tractable bacteria.

Bacteria produce secreted compounds that have the potential to affect the physiology of their microbial neighbors. Here we describe a coculture screen that allows detection of such chemically mediated interspecies interactions by mixing soil microbes with fluorescent transcriptional reporter strains of Bacillus subtilis on solid media.

화학적 매개 종간의 상호 작용을 검출하는 공 배양을 사용하여

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by ...

생물학

Fluorescent Reporter 정량화를 통한 전체 장기 병원체 추적

High&#45;Resolution Three&#45;Dimensional Whole&#45;Organ Tomography of Microbial Infections

Most infections take place within three-dimensional host tissues with intricate anatomy and locally varying host physiology. The positioning of pathogen cells within this diverse environment significantly affects their stress levels, responses, fate, and contribution to the overall progression of the disease and treatment failure. However, due to the technical difficulties in locating &#181;m-sized pathogen cells within cm-sized host organs, this area of research has been relatively unexplored. Here, we present a method for addressing this challenge. We employ serial two-photon tomography and AI-enhanced image analysis to locate individual Salmonella cells throughout the entire spleen, liver lobes, and whole lymph nodes of infected mice. Using fluorescent reporters and in vivo antibody administration, the replication rate of single Salmonella cells, their local interaction with specific immune cells, and bacterial responses to antibiotics can be determined. These methodologies open avenues for a comprehensive examination of infections, their prevention, and treatment within the three-dimensional tissue context.

저자 스포트라이트: Unraveling Bacterial Responses to Antibiotics and Immune System in Tissues(조직의 항생제 및 면역 체계에 대한 박테리아 반응 규명)

Here, we describe a procedure that enables organ-wide detection of pathogenic bacteria during infection and quantification of fluorescent reporter activities.

고분해능 3차원 전장기 단층촬영(High-resolution three-dimensional whole-organ tomography of microbial infections)

Most infections take place within three-dimensional host tissues with intricate anatomy and locally varying host physiology. The positioning of pathogen ...

카테고리의 감지 세균성 병원균에 대한 '발광 생물'리포터 파지

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