Name: utilizing the ethylene-releasing compound, 2-chloroethylphosphonic acid, as a tool to study ethylene response in bacteria
Uploaded: 2016-11-10T14:00:00
Description: Watch this Scientific Journal Video about Utilizing the Ethylene-releasing Compound, 2-Chloroethylphosphonic Acid, as a Tool to Study Ethylene Response in Bacteria at JoVE.com

The authors thank Dr. Dario Bonetta for providing Arabidopsis thaliana seeds and for technical assistance in regards to the triple response assay, as well as Simone Quaranta for help with FT-IR. This work was supported by a Natural Sciences and Engineering Research Council of Canada Discovery Grant (NSERC-DG) to JLS, an Ontario Graduate Scholarship (OGS) to RVA, and a Queen Elizabeth II Graduate Scholarship in Science and Technology (QEII-GSST) to AJV.

1. 화학 <ol><li> 500 밀리미터의 NaH 2 PO 4 · 산성화의 H 2 O (137.99 g / mol)의 (PH 500 mM의 CEPA (144.49 g / 몰)의 용액에, 두 500 mM의 염화나트륨 (58.44 g / 몰)로 이루어진 용액을 준비 2.5) 초순수 0.1 N 염산. 솔루션 분명히 때까지 소용돌이를 사용하여 섞는다. </li><li> 직렬 5 mM 내지 50 mM의 축적량을 획득하기 위해 동일한 용매 (10 배), 500 mM의 용액을 희석. </li><li> 초순수의 1-aminocyclopropane ?...

<ol>
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여기에 설명 된 방법은 모델 생물, K.를 사용하여 세균 에틸렌 반응의 연구 CEPA에서 에틸렌의 생산 현장에서 개요 xylinus. 에틸렌의 pH를 CEPA 순수한 에틸렌 가스 또는 전문 실험실 장비에 대한 필요성을 부정하는 이상으로 3.5 10,11이있는 수성 매체를 보충함으로써 제조 할 수 있으므로이 방법은 매우 유용하다. 이 방법은 세균에서 유래 CEPA 에틸렌의 효과를 연구에 국한?...

올레핀 에틸렌 (C 2 H 4)는 석탄 가스 램프를 사용하여 실험실에서 성장 완두콩 모종, (배축)이 짧았다 유래 된 비정상적인 형태를 나타낸다 관찰되었을 때 제 1901 식물 호르몬으로 발견 두꺼운 정상 완두콩 모종에 비해 옆으로 구부러진; 표현형 나중에 트리플 응답 1,2라고. 후속 연구는 에틸렌은 성장, 스트레스 반응, 과일 숙성 및 노화 3. 애기 장대 식물 생물학 연구를위한 모델 생물로 다수의 발달 과정을 조절하는 중요한 phytohormone가 잘 에틸렌에 대한 응답에 관해서 연구되어왔다 있음을 보여 주었다. 몇몇 에틸렌 반응 돌연변이 어두운 자란 A. 관찰 트리플 반응 표현형을 이용하여 분리 된 에틸렌 1,4,5의 존재 장대 모종. 식물에서 에틸렌 생합성 제조를위한 전구체는 1-A 인minocyclopropane 카복실산 (ACC) (6)과 공통 트리플 응답 표현형 1,4,5 리드 내생 에틸렌 생산을 증가시키는 트리플 응답 분석시 사용된다. 에틸렌 반응은 식물에 널리 연구되고 있지만, 세균에 외인성 에틸렌의 효과는 크게 식물 세균의 확대에도 불구 파악 하였다 연관된다. 한 연구는 특정 슈도모나스 균주는 탄소와 에너지 (7)의 유일한 소스로 에틸렌을 사용하여 살아남을 수 있다고보고했다. 그러나,이 연구는 박테리아가 에틸렌에 반응 것을 증명하고있다. 첫 번째 연구는 보여 주었다 녹농균, P.의 균주 fluorescens, P. 푸티 및 P. syringae 용융 아가 순수한 에틸렌 가스 (8) 평형화 화성 완충액과 혼합 한 아가 플러그 어 세이를 사용하여 에틸렌을 향해 주 화성이었다. 그러나, 우리의 지식, 더 푸르트는 없었다순수한 에틸렌 가스를 사용하여 응급실 보고서 때문에 특별한 장비없이 실험실 가스 운반의 성사 때문일 세균 에틸렌 응답을 특성화한다. 세균 에틸렌 반응의 두번째 보고서 에틸렌 9 xylinus 과일 관련 세균 Komagataeibacter (이전 Gluconacetobacter)에서 박테리아 셀룰로오스 생산 영향 유전자 발현이 증가하는 것이 보였다. 이 경우에, 에틸렌 - 방출 화합물, 2- 클로로 에틸 포스 폰산 (에 CEPA)은 순수한 에틸렌 가스 또는 특수 장비에 대한 필요성을 우회하여 박테리아 성장 배지 내에서 동일계 에틸렌을 제조 하였다. - 14 염기 - 촉매, 일차 반응 pH를 3.5 내지 12 10,11 전술 한 몰 비율은 1 CEPA 에틸렌을 생성한다. CEPA의 열화가 긍정적 에틸의 제조에서 pH와 온도를 13, 14 및 결과와 상호 관련엔, 염화 인산염. CEPA는 기체 에틸렌 편리한 대안 에틸렌 박테리아 응답을 공부에 관심있는 연구자를 제공합니다. 다음 프로토콜의 전반적인 목적은 박테리아의 에틸렌 반응을 연구하는 간단하고 효율적인 방법을 제공하는 박테리아 성장 배지의 CEPA 분해로부터 에틸렌 생산의 생리 학적 관련성 레벨 검증을 포함하는, 배양 pH를 분석 CEPA 분해가 동안 손상되지 않게하려면 세균의 성장 및 박테리아 형태 및 표현형에 에틸렌의 효과 평가. 우리는 K.을 사용하여 이러한 프로토콜을 보여 xylinus 그러나, 이러한 프로토콜들은 적절한 성장 배지를 사용하여 표현형 분석하여 다른 세균에서 에틸렌 반응을 연구하기 위해 적응 될 수있다.

<table border="0" cellpadding="0" cellspacing="0" width="1018">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">1-aminocyclopropane carboxylic acid (ACC)</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:119px;">A3903</td>
			<td style="width:432px;">Biosynthetic precursor of ethylene in plants</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">4-sector Petri dish</td>
			<td style="width:136px;">Phoenix Biomedical</td>
			<td style="width:119px;">CA73370-022</td>
			<td>For testing triple response</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Agar</td>
			<td style="width:136px;">BioShop</td>
			<td style="width:119px;">AGR001.1</td>
			<td>To solidify medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Canon Rebel T1i DLSR camera</td>
			<td style="width:136px;">Canon</td>
			<td style="width:119px;">3818B004</td>
			<td>For pictures of pellicles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Cellulase from Trichoderma reesei ATCC 26921&#160;</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:119px;">C2730</td>
			<td>Aqueous solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Citric acid</td>
			<td style="width:136px;">BioShop</td>
			<td style="width:119px;">CIT002.500</td>
			<td>For SH medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Commercial bleach</td>
			<td style="width:136px;">Life Brand</td>
			<td style="width:119px;">57800861874</td>
			<td>Bleach for seed sterilization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Concentrated HCl</td>
			<td style="width:136px;">BioShop</td>
			<td style="width:119px;">HCL666.500</td>
			<td>Hydrochloric acid for pH adjustment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Digital USB microscope</td>
			<td style="width:136px;">Plugable</td>
			<td style="width:119px;">N/A</td>
			<td>For pictures of colonies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Ethephon (&#8805; 96%; 2-chloroethylphosphonic acid)</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:119px;">C0143</td>
			<td>Ethylene-releasing compound</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Glucose</td>
			<td style="width:136px;">BioBasic</td>
			<td style="width:119px;">GB0219</td>
			<td>For SH medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Komagataeibacter xylinus ATCC 53582</td>
			<td style="width:136px;">ATCC</td>
			<td style="width:119px;">53582</td>
			<td>Bacterial cellulose-producing alphaproteobacterium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Microcentrifuge tube</td>
			<td style="width:136px;">LifeGene</td>
			<td style="width:119px;">LMCT1.7B</td>
			<td>1.7 mL microcentrifuge tube</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Murashige and Skoog (MS) basal medium&#160;</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:119px;">M5519</td>
			<td>Arabidopsis thaliana growth medium</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:331px;">Na2HPO4&#183;7H2O&#160;</td>
			<td style="width:136px;">BioShop</td>
			<td style="width:119px;">SPD579.500</td>
			<td>Sodium phosphate, dibasic heptahydrate for SH medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">NaCl</td>
			<td style="width:136px;">BioBasic</td>
			<td style="width:119px;">SOD001.1</td>
			<td>Sodium chloride for saline and control solution</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:331px;">NaH2PO4&#183;H2O&#160;</td>
			<td style="width:136px;">BioShop</td>
			<td style="width:119px;">SPM306.500</td>
			<td>Sodium phosphate, monobasic monohydrate for control solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">NaOH</td>
			<td style="width:136px;">BioShop</td>
			<td style="width:119px;">SHY700.500</td>
			<td>Sodium hydroxide for pH adjustment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Paraffin film</td>
			<td style="width:136px;">Parafilm</td>
			<td style="width:119px;">PM996</td>
			<td>For sealing plates and flasks</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Peptone (bacteriological)</td>
			<td style="width:136px;">BioShop</td>
			<td style="width:119px;">PEP403.1</td>
			<td>For SH medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Petroff-Hausser counting chamber</td>
			<td style="width:136px;">Hausser scientific</td>
			<td style="width:119px;">3900</td>
			<td>Bacterial cell counting chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Polyethersulfone sterilization filter 0.2 &#181;m</td>
			<td style="width:136px;">VWR</td>
			<td style="width:119px;">28145-501</td>
			<td>For sterilizing cellulase</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Sucrose</td>
			<td style="width:136px;">BioShop</td>
			<td style="width:119px;">SUC600.1</td>
			<td>Sucrose for MS medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Yeast extract</td>
			<td style="width:136px;">BioBasic</td>
			<td style="width:119px;">G0961</td>
			<td>For SH medium</td>
		</tr>
	</tbody>
</table>

utilizing the ethylene-releasing compound, 2-chloroethylphosphonic acid, as a tool to study ethylene response in bacteria

Ethylene (C2H4) is a gaseous phytohormone that is involved in numerous aspects of plant development, playing a dominant role in senescence and fruit ripening. Exogenous ethylene applied during early plant development triggers the triple response phenotype; a shorter and thicker hypocotyl with an exaggerated apical hook. Despite the intimate relationship between plants and bacteria, the effect of exogenous ethylene on bacteria has been greatly overlooked. This is partly due to the difficulty of controlling gaseous ethylene within the laboratory without specialized equipment. 2-Chloroethylphosphonic acid (CEPA) is a compound that decomposes into ethylene, chlorine, and phosphate in a 1:1:1:1 molar ratio when dissolved in an aqueous medium of pH 3.5 or greater. Here we describe the use of CEPA to produce in situ ethylene for the investigation of ethylene response in bacteria using the fruit-associated, cellulose-producing bacterium Komagataeibacter xylinus as a model organism. The protocols described herein include both the verification of ethylene production from CEPA via the Arabidopsis thaliana triple response assay and the effects of exogenous ethylene on K. xylinus cellulose production, pellicle properties and colonial morphology. These protocols can be adapted to examine the effect of ethylene on other microbes using appropriate growth media and phenotype analyses. The use of CEPA provides researchers with a simple and efficient alternative to pure ethylene gas for the routine determination of bacterial ethylene response.

트리플 응답 분석하여 SH 배지에서 CEPA에서 에틸렌 해방 (PH 7)의 검증을위한 개략도 판 설치는 그림 1A에 표시됩니다 - C를. 펠리클 프로토콜을 도시하는 흐름도는도 2 다크 성장한 A. 같다 장대 모종 ACC의 존재 및 SH 매체 CEPA의 분해를 통해 제조 된 에틸렌 (pH가 7)의 존재하에 삼중 반응 표현형 (과장 정점 후크 짧고 두꺼운 ?...

Watch this Scientific Journal Video about Utilizing the Ethylene-releasing Compound, 2-Chloroethylphosphonic Acid, as a Tool to Study Ethylene Response in Bacteria at JoVE.com

Utilizing the Ethylene-releasing Compound, 2-Chloroethylphosphonic Acid, as a Tool to Study Ethylene Response in Bacteria

The protocols outlined herein facilitate the convenient investigation of bacterial ethylene responses by utilizing 2-chloroethylphosphonic acid (CEPA). Ethylene is produced in situ through the decomposition of CEPA in an aqueous bacterial growth medium, circumventing the requirement for pure ethylene gas.

도구로 에틸렌 방출 화합물, 2- 클로로 에틸 산을 활용하여 박테리아의 에틸렌 응답을 공부하기

Ethylene (C2H4) is a gaseous phytohormone that is involved in numerous aspects of plant development, playing a dominant role in senescence and fruit ...

Research

JoVE Journal

Biochemistry

Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions

단백질 - 단백질 상호 작용을 연구하는 도구로 형광 이방성

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is ...

연구

생화학

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

항온, 발현, 정제, 인간 세로토닌 전달체 결정화 바운드<em&gt; S</em&gt; 시탈 로프 람

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug ...

Titration ELISA as a Method to Determine the Dissociation Constant of Receptor Ligand Interaction

적정 수용 체 Ligand 상호 작용의 분리 상수를 결정 하는 방법으로 ELISA

The dissociation constant describes the interaction between two partners in the binding equilibrium and is a measure of their affinity. It is a crucial ...

A Colorimetric Method for Measuring Iron Content in Plants

식물에 철 분 함량을 측정 하는 색도계 방법

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

소규모 색도계 분석 실험의 세포내 젖이 나올와 꼬마 선 충 의 선 충 Pyruvate

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

인지질 단백질 Annexin a 2 석 영 공명 기를 사용 하 여 지원 되는 고체 지질 Bilayers에 바인딩의 바인딩 역학 공부 되는 낭비 적인 Microgravimetry

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

Isolation of Lipoprotein Particles from Chicken Egg Yolk for the Study of Bacterial Pathogen Fatty Acid Incorporation into Membrane Phospholipids

세균성 병원체 지방산 을 막 인지질로 통합하는 연구를 위해 닭 계란 노른자에서 지단백질 입자의 분리

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the ...

Activated Cross-linked Agarose for the Rapid Development of Affinity Chromatography Resins - Antibody Capture as a Case Study

선호도 크로마토그래피 수지의 신속한 개발을 위한 활성화된 상호 연결 아가로스 - 사례 연구로서의 항체 포획

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall ...

In Vivo Hydroxyl Radical Protein Footprinting for the Study of Protein Interactions in Caenorhabditis elegans

In Vivo Hydroxyl Radical Protein Footprinting for the Study of Protein Interactions in Caenorhabditis elegans

체노하브디염 예쁜꼬마선충의 단백질 상호작용 연구를 위한 생체내 하이드록실 라디칼 단백질 발자국

Fast oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting (HRPF) method used to study protein structure, protein-ligand interactions, ...

Utilizing Time-Resolved Protein-Induced Fluorescence Enhancement to Identify Stable Local Conformations One &#945;-Synuclein Monomer at a Time

Utilizing Time-Resolved Protein-Induced Fluorescence Enhancement to Identify Stable Local Conformations One ÃƒÅ½Ã‚Â±-Synuclein Monomer at a Time

시간 해결 된 단백질 유발 형광 향상을 활용하여 안정적인 지역 적합성을 식별하기 위해 한 번에 한 α 시뉴클레인 단백기를 식별합니다.

Using spectroscopic rulers to track multiple conformations of single biomolecules and their dynamics have revolutionized the understanding of structural ...