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Immunology and Infection

用于量化Vibrio Fischeri分离物之间竞争交互的联合孵化测定

Published: July 22nd, 2019

DOI:

10.3791/59759

1Department of Marine Sciences, University of North Carolina, Chapel Hill

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。

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。

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

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1. 为联合孵化准备菌株

  1. 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。
  2. 确定将采用哪些选择和筛选方法来区分共育中的分离物
    1. 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?.......

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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?.......

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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。

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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。

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NameCompanyCatalog NumberComments
1.5 mL Microcentrifuge TubesFisher05-408-129
10 μL multichannel pipette
100 μL multichannel pipette
300 μL multichannel pipette
10 μL single channel pipette
20 μL single channel pipette
200 μL single channel pipette
1000 μL single channel pipette
24-well platesFisher07-200-84sterile with lid
96-well platesVWR10062-900sterile with lid
Calculator
ChloramphenicolSigmaC0378stock (20 mg/mL in Ethanol); final concentration in media (2 μg /mL LBS)
Fluorescence dissecting microscope with camera and imaging software
forcepsFisher08-880
Kanamycin SulfateFisherBP906-5stock (100 mg/mL in water, filter sterilize); final concentration in media (1 μg/mL LBS)
Nitrocellulose membrane (FS MCE, 25MM, NS)FisherSA1J788H50.22 μm nitrocellulose membrane (pk of 100)
petri platesFisherFB0875713sterile with lid
Spectrophotometer
Semi-micro cuvettesVWR97000-586
TipOne 0.1-10 μL starter systemUSA Scientific1111-350010 racks
TipOne 200 μL starter systemUSA Scientific1111-50010 racks
TipOne 1000 μL starter systemUSA Scientific1111-252010 racks
Vortex
NameCompanyCatalog NumberComments
LBS media
1M Tris Buffer (pH ~7.5)50 mL 1 M stock buffer (62 mL HCl, 938 mL DI water, 121 g Trizma Base)
Agar TechnicalFisherDF0812-17-915 g (Add only for plates)
DI water950 mL
Sodium ChlorideFisherS640-320 g
TryptoneFisherBP9726510 g
Yeast ExtractFisherBP9727-25 g

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