Name: coincubation assay for quantifying competitive interactions between vibrio fischeri isolates
Uploaded: 2019-07-22T13:00:00
Description: Watch this Scientific Journal Video about Coincubation Assay for Quantifying Competitive Interactions between Vibrio fischeri Isolates at JoVE.com

我们要感谢审阅者提供的有用反馈。A.N.S.由戈登和贝蒂·摩尔基金会通过授予GBMF 255.03向生命科学研究基金会提供支持。

1. 为联合孵化准备菌株
<ol><li>选择适当的参考菌株,作为联合孵育测定期间细菌竞争的目标。请参阅讨论,了解选择参考应变时的最佳做法以及参考应变将如何影响结果。在此协议中,V.菲舍尔应变 ES114 将作为参考应变。</li>
	<li>
确定将采用哪些选择和筛选方法来区分共育中的分离物
	<ol>
<li>通常,使用含有不同抗生素耐药性基因(如卡那霉素或氯霉素)的稳定质粒来转?...

<ol>
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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rules+of+engagement:+the+type+VI+secretion+system+in+Vibrio+cholerae.">Rules of engagement: the type VI secretion system in Vibrio cholerae.</a> Trends in microbiology. 25 (4), 267-279 (2017).</li><li>Speare, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+symbionts+use+a+type+VI+secretion+system+to+eliminate+competitors+in+their+natural+host.">Bacterial symbionts use a type VI secretion system to eliminate competitors in their natural host.</a> Proceedings of the National Academy of Sciences of the United States of America. 115 (36), E8528-E8537 (2018).</li><li>Shank, E. 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上述共育测定为发现细菌间竞争提供了一种强有力的方法。这种方法可以识别V.fischeri分离和描述竞争机制之间的特定竞争19。虽然所述方法针对海洋细菌V.菲舍尔进行了优化,但可以轻松修改,以适应其他细菌种类,包括临床和环境分离物。需要注意的是,竞争机制通常有条件地受到5、6、23、24、25、26、27的制约。<sup c...

本手稿概述了一种基于培养的方法,用于确定两种细菌分离物是否具有竞争性相互作用。在研究混合种群时,评估细菌分离物相互作用的程度非常重要,特别是分离物是否通过干扰机制直接竞争。干扰竞争是指一个人口直接抑制增长或杀死竞争对手1的相互作用。这些相互作用是重要的识别,因为它们可以有深刻的影响微生物群落的结构和功能2,3。
微生物竞争机制已广泛发现来自不同环境的细菌基因组,包括宿主相关细菌和自由生存细菌4、5、6、7 8,9.已经描述了各种竞争策略10,11包括可扩散的机制,如杀菌化学品1,12和分泌抗菌肽13,以及需要细胞-细胞接触将抑制作用剂转移到目标细胞9、14、15、16、17的接触依赖机制,18.
虽然基于培养的共育物在微生物学5、8、19中常用,但本手稿概述了如何使用测定来描述竞争机制,以及适应与其他细菌物种一起使用的协议。此外,此方法还描述了分析和呈现数据的多种方法,以回答有关竞争交互性质的不同问题。虽然本文所述技术以前曾用于识别共生Vibrio Fischeri细菌19共生菌株之间特定竞争背后的细菌间杀灭机制,但它们适用于许多细菌物种,包括环境分离物和人类病原体,可用于评估接触依赖性和可扩散的竞争机制。协议中的步骤可能需要优化其他细菌种类。鉴于更多的模型系统正在扩大他们的研究,从同源生物的使用,包括不同的基因型10,16,20,21,这种方法将是一个宝贵的资源寻求了解竞争如何影响多菌株或多物种系统的研究人员。

<table>
	<tbody>
		<tr>
			<td>1.5 mL Microcentrifuge Tubes</td>
			<td>Fisher</td>
			<td>05-408-129</td>
			<td></td>
		</tr>
		<tr>
			<td>10 &#956;L multichannel pipette</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#956;L multichannel pipette</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>300 &#956;L multichannel pipette</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10 &#956;L single channel pipette</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>20 &#956;L single channel pipette</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#956;L single channel pipette</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1000 &#956;L single channel pipette</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plates</td>
			<td>Fisher</td>
			<td>07-200-84</td>
			<td>sterile with lid</td>
		</tr>
		<tr>
			<td>96-well plates</td>
			<td>VWR</td>
			<td>10062-900</td>
			<td>sterile with lid</td>
		</tr>
		<tr>
			<td>Calculator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Sigma</td>
			<td>C0378</td>
			<td>stock (20 mg/mL in Ethanol); final concentration in media (2 &#956;g /mL LBS)</td>
		</tr>
		<tr>
			<td>Fluorescence dissecting microscope with camera and imaging software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>forceps</td>
			<td>Fisher</td>
			<td>08-880</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin Sulfate</td>
			<td>Fisher</td>
			<td>BP906-5</td>
			<td>stock (100 mg/mL in water, filter sterilize); final concentration in media (1 &#956;g/mL LBS)</td>
		</tr>
		<tr>
			<td>Nitrocellulose membrane (FS MCE, 25MM, NS)</td>
			<td>Fisher</td>
			<td>SA1J788H5</td>
			<td>0.22 &#956;m nitrocellulose membrane (pk of 100)</td>
		</tr>
		<tr>
			<td>petri plates</td>
			<td>Fisher</td>
			<td>FB0875713</td>
			<td>sterile with lid</td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Semi-micro cuvettes</td>
			<td>VWR</td>
			<td>97000-586</td>
			<td></td>
		</tr>
		<tr>
			<td>TipOne 0.1-10 &#956;L starter system</td>
			<td>USA Scientific</td>
			<td>1111-3500</td>
			<td>10 racks</td>
		</tr>
		<tr>
			<td>TipOne 200 &#956;L starter system</td>
			<td>USA Scientific</td>
			<td>1111-500</td>
			<td>10 racks</td>
		</tr>
		<tr>
			<td>TipOne 1000 &#956;L starter system</td>
			<td>USA Scientific</td>
			<td>1111-2520</td>
			<td>10 racks</td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>LBS media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1M Tris Buffer (pH ~7.5)</td>
			<td></td>
			<td></td>
			<td>50 mL 1 M stock buffer (62 mL HCl, 938 mL DI water, 121 g Trizma Base)</td>
		</tr>
		<tr>
			<td>Agar Technical</td>
			<td>Fisher</td>
			<td>DF0812-17-9</td>
			<td>15 g (Add only for plates)</td>
		</tr>
		<tr>
			<td>DI water</td>
			<td></td>
			<td></td>
			<td>950 mL</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher</td>
			<td>S640-3</td>
			<td>20 g</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Fisher</td>
			<td>BP97265</td>
			<td>10 g</td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>Fisher</td>
			<td>BP9727-2</td>
			<td>5 g</td>
		</tr>
	</tbody>
</table>

coincubation assay for quantifying competitive interactions between vibrio fischeri isolates

本手稿描述了一种基于培养的共生测定,用于检测和描述两种细菌群体之间的竞争相互作用。该方法采用稳定的质粒,使每个种群分别具有不同的抗生素抗性能力和荧光蛋白,用于选择和视觉鉴别。在这里,我们描述了竞争维布里奥菲舍尔菌株的制备和联合孵化、荧光显微镜成像和定量数据分析。这种方法很简单,产生快速的结果,并可用于确定一个种群是杀死还是抑制另一个种群的生长,以及竞争是通过可扩散的分子进行中介的,还是需要直接的细胞-细胞接触。由于每个细菌群体表达不同的荧光蛋白,因此测定允许在混合菌群中对相互竞争的人群进行空间歧视。虽然所述方法使用共生细菌V. 菲舍尔使用针对该物种优化的条件执行,但该协议可以适用于大多数可培养的细菌分离物。

为了评估细菌群之间的竞争相互作用,为V.fischeri开发并优化了联合孵育测定方案。该方法利用编码抗生素抗性基因和荧光蛋白的稳定质粒,允许每个菌株的微分选择和视觉鉴别。通过分析从共孵化测定中收集的数据,可以识别相互作用的竞争结果和相互作用的机制。例如,使用V.菲舍尔基分离物进行了以下实验。凝结菌株含有两个稳定的质粒之一:pVSV102表示卡那霉?...

Watch this Scientific Journal Video about Coincubation Assay for Quantifying Competitive Interactions between Vibrio fischeri Isolates at JoVE.com

Coincubation Assay for Quantifying Competitive Interactions between Vibrio fischeri Isolates

细菌编码各种机制,参与细菌间竞争。在这里,我们提出了一个基于培养的协议,用于描述细菌分离物之间的竞争相互作用,以及它们如何影响混合种群的空间结构。

用于量化Vibrio Fischeri分离物之间竞争交互的联合孵化测定

This manuscript describes a culture-based, coincubation assay for detecting and characterizing competitive interactions between two bacterial populations. ...

我们的协议提供了一种简单而稳健的方法，用于识别和描述不同菌株的 Vibrio 菲舍尔细菌之间的竞争机制。此方法使用稳定的质粒编码不同的抗生素耐药基因和荧光蛋白，允许差异选择和视觉识别每个细菌菌株在混合培养。首先，使用无菌牙签从一粒大米大小的阿加板中刮出细胞。将细胞重新淹没在含有1毫升LBS汤的1.5毫升微离心管中。要分解细胞团，请将它们压入管的一侧，然后大力上下移液器。关闭管和涡流一到两秒钟，如果细胞团仍然可见，重复，直到样品在视觉上均匀。对所有样品重复这些制备步骤，从细胞刮擦到涡旋。测量并记录所有样品的光学密度为 600 纳米。为了获得准确的 OD 测量，使用 LBS 汤将样品稀释多达 10 倍。然后将每个样品规范化为所需的 OD 600。要将参考应变和竞争对手应变以一比一的比例混合，将每株100微升的亚光升加到OD 1.0中，加入一个标有1.5毫升的离心管和涡流，一至两秒钟。作为控件，将参考应变与本身的差分标记版本和涡流混合。实现正确的起始比至关重要，因为它可以显著改变实验的结果。为了确保成功，此步骤可能需要对给定细菌物种进行优化。在每个生物复制重复此步骤后，应有四个生物复制，以不同标记参考菌株作为对子，四个生物复制与差分标记参考菌株和竞争对手菌株。为了制作孵化后用于荧光显微镜的培养点，在含有LBS agar的培养板上加入10个每个控制和实验混合物的10微升。斑点在长凳上完全干燥后，在24摄氏度下孵育板24小时。为了在实验结束时，将培养点用于量化每个菌株的聚落形成单位，将每个对照装置和实验混合物的10微升添加到24个井板中，每孔含有1毫升LBS汞。斑点完全干燥后，在24摄氏度下孵育板5小时。要连续稀释起始开库混合物，请旋转 96 井板 90 度，以便有 8 列和 12 行。用应变 ID、复制编号和以 t 零开始的时间标记每行。给LBS加糖板贴上标签，并辅以适当的抗生素，以发现稀释系列，同时确保确定每个抗生素板选择的菌株。使用多通道移液器，将 180 微升 LBS 汤添加到每条井中，使第一柱为未稀释的硬币分配混合物留空。使用 200 微升单通道移液器，将 100 微升的硬币孵化混合物从管转移到第一柱的第一个井，然后丢弃尖端，对所有开投混合物重复。使用多通道移液器，将 20 微升从第一列转移到第二列，然后上下调液器进行混合。丢弃提示，重复每列，同时与每次稀释的上下移液数保持一致。最后，每行将包含初始混合物的 10 倍串行稀释。使用多通道移液器，配备八个吸头，在稀释系列中从每一井吸出五微升，并点到卡纳霉素补充的 LBS 加糖板上，该板可选择参考应变。重复此步骤，为竞争对手选择在氯霉素补充板上发现应变。斑点完全干燥后，将板放在24摄氏度的培养箱中。在硬币孵化点和24个井板在24摄氏度下孵育5小时后，在每孔中加入1毫升LBS汤。通过上下移液，直到所有细胞被重新注入，与细胞在所有复制中重新注入的活力保持一致。一旦每个开库点重新暂停，准备一个96井板的连续稀释和LBS加糖板与适当的抗生素，以发现稀释的方式与以前一样。要完成 24 孔板中 t 最终测量的串行稀释，请执行用于连续稀释起始氧化铝的步骤，并像之前一样在 LBS 加糖板上点点每次稀释。继续成像培养板上的硬币点，并进行数据分析，如手稿中所述。在实验治疗中，参考菌株和竞争对手菌株在实验的开始和结束时与控制治疗一致的比例为0.5。Log RCI 值在统计上与控制处理的零没有差异，或者当参考应变与竞争对手菌株一一孵育时。这些数据表明，参考菌株与竞争对手菌株之间没有竞争。相比之下，当参考应变与竞争对手应变2一起孵育时，参考应变从开始时的0.5比例降至实验结束时小于0.01的比例。此外，当参考应变与竞争对手应变2一起孵育时，日志RCI值明显大于零。总之，这些数据表明，在存在竞争对手菌株的情况下，参考菌株的比例下降，这表明菌株之间的竞争。当参考菌株与竞争对手菌株1一孵育时，两种菌株的C双在5小时内增加，且没有明显不同，表明没有竞争发生。此外，观察到大约 3200% 的恢复，与控件在统计学上没有差异。然而，当参考应变与竞争对手应变2一起孵育时，参考应变CU在五小时内明显低于控制中的2个CFO应变。此外，观察到大约4%的恢复率明显低于控制。总之，这些数据表明，应变2出竞争参考株通过杀死其细胞。此过程中最重要的步骤是实现正确的起始比率，因为它可以显著影响结果。

Research

JoVE Journal

Immunology and Infection

Coincubation Assay for Quantifying Competitive Interactions between Vibrio fischeri Isolates

Colonization of Euprymna scolopes Squid by Vibrio fischeri

Colonization of Euprymna scolopes Squid by Vibrio fischeri

定植 Euprymna scolopes鱿鱼由弧菌鲵</em

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no ...

科研

免疫与感染

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

监测树突状细胞迁移<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H磁共振成像

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

一种新型<em&gt;在体外</em&gt;模型研究人类全血细胞间的相互作用

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways ...

Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions

在剪切粘结力的测定T淋巴细胞粘附分子相互作用的研究

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

RNA的疗效和毒性的评价基因或药物治疗靶向HIV-1的生产使用

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

对于检测到的敏感性和特异性显色琼脂培养基发展<em&gt;副溶血性弧菌</em&gt;及其他<em&gt;弧菌</em&gt;物种

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the ...

Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

用于维持和区分生物型的实验室技术<em&gt;霍乱弧菌</em&gt;临床和环境隔离

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global ...

Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells

Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells

诱导细胞分化和单细胞成像<em&gt;副溶血性弧菌</em游泳者和Swarmer细胞

The ability to study the intracellular localization of proteins is essential for the understanding of many cellular processes. In turn, this requires the ...

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

体内人淋巴组织和女性生殖器粘膜与人体免疫机能丧失病毒1和 Histoculture 的感染

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under ...

Quantifying Vibrio cholerae Colonization and Diarrhea in the Adult Zebrafish Model

Quantifying Vibrio cholerae Colonization and Diarrhea in the Adult Zebrafish Model

成年斑马鱼模型中霍乱弧菌定植与腹泻的量化研究

Vibrio cholerae is best known as the infectious agent that causes the human disease cholera. Outside the human host, V. cholerae primarily exists in the ...