Name: single-cell analysis of bacillus subtilis biofilms using fluorescence microscopy and flow cytometry
Uploaded: 2012-02-15T12:00:00
Description: Watch this Scientific Journal Video about Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry at JoVE.com

Diese Arbeit wird von den Young Investigator Research Program gefördert, vom Zentrum für Infektionsforschung (zinf) von der Universität Würzburg. Juan Garcia-C Betancur ist ein Doktorand von der Graduate School of Life Sciences (GSLS) der Universität Würzburg.

1. Labeling B. subtilis und Biofilm-Assay <ol><li> Amplify von PCR, das die Promotorregion des Gens von Interesse. Wir zeigen, wie beispielsweise das Klonen von P Tapa, der Promotor der Gene, die für die Herstellung von Tasa Matrixprotein 26. Klon P Tapa in pkm008 Vektor (erstellt von der Rudner Labor, Harvard Medical School. Boston, USA) (Abb. 2). </li><li> Linearisieren die Plasmide durch enzymatische Verdauung (Enzyme empfohlen, XhoI). </li><li> Induziere...

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Die Tatsache, dass bakterielle Gemeinschaften Subpopulationen von Zellen, die bestimmte Gruppe von Genen, beweist die Komplexität mikrobieller Gemeinschaften 33,34 zeigen. Dieses Protokoll soll helfen, festzustellen, ob die Expression eines beliebigen Gens von Interesse für eine bestimmte Subpopulation von spezialisierten Zellen innerhalb der mikrobiellen Gemeinschaft beschränkt. Die Visualisierung dieser Subpopulationen erfordert die Entwicklung neuer Techniken, da herkömmliche Methoden Genexpression ode...

<table border="1"><tbody><tr><td> Technik </td><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td></tr><tr><td rowspan="11"> MSgg Zusammensetzung </td><td> 5 mM Kaliumphosphat </td><td> Roth </td><td> 6878 </td></tr><tr><td> 100 mM MOPS </td><td> Sigma-Aldrich </td><td> M1254 </td></tr><tr><td> 2 mM Magnesiumchlorid </td><td> Roth </td><td> 2189,1 </td></tr><tr><td> Calciumchlorid 700μM </td><td> Roth </td><td> A119.1 </td></tr><tr><td> Eisenchlorid 50 uM </td><td> Sigma-Aldrich </td><td> 157740 </td></tr><tr><td> Zinkchlorid 1 &amp;mgr; M </td><td> Applichem </td><td> A2076 </td></tr><tr><td> Thiamin 2 &amp;mgr; m </td><td> Sigma-Aldrich </td><td> 74625 </td></tr><tr><td> Glycerin 0,5% </td><td> Roth </td><td> 7533 </td></tr><tr><td> GlutaMate 0,5% </td><td> Sigma-Aldrich </td><td> 49621 </td></tr><tr><td> Tryptophan 50μg/ml </td><td> Sigma-Aldrich </td><td> T0254 </td></tr><tr><td> Phenylalanin 50μg/ml </td><td> Sigma-Aldrich </td><td> P2126 </td></tr><tr><td> Zellfixierung </td><td> Paraformaldehyd </td><td> Roth </td><td> 0335 </td></tr><tr><td></td><td> Bezeichnung des Geräts </td><td> Firma </td><td> Katalog-Nummer </td></tr><tr><td> Ultraschallbehandlung </td><td> Zelle Sonicator </td><td> Bandelin </td><td> D-1000 </td></tr><tr><td> Fluoreszenz-Mikroskopie </td><td> Fluoreszenz-Mikroskop </td><td> Leica </td><td> DMI6000B </td></tr><tr><td></td><td> Name der Software </td><td> Firma </td><td> Katalog-Zahl </td></tr><tr><td> Fluoreszenz-Mikroskopie </td> &lt;td&gt; Asaf </td><td> Leica </td><td></td></tr><tr><td> Die Durchflusszytometrie </td><td> FCASDiva </td><td> BD </td><td></td></tr><tr><td> Die Durchflusszytometrie </td><td> FlowJo </td><td> Treestar </td><td></td></tr></tbody></table>

single-cell analysis of bacillus subtilis biofilms using fluorescence microscopy and flow cytometry

Biofilm-Bildung ist ein allgemeines Attribut auf fast allen Bakterien 6.1. Wenn Bakterien Biofilme zu bilden, sind Zellen, in der extrazellulären Matrix, die hauptsächlich gebildet wird von Proteinen und Exopolysacchariden, neben anderen Faktoren 7-10 umhüllt. Die mikrobielle eingeschlossen innerhalb der Biofilme häufig zeigt die Differenzierung von unterschiedlichen Subpopulation von spezialisierten Zellen 11-17. Diese Subpopulationen koexistieren und zeigen oft räumliche und zeitliche Organisation innerhalb des Biofilms 18-21. Biofilm-Bildung in den Modellorganismus Bacillus subtilis erfordert die Differenzierung von Subpopulationen von spezialisierten Zellen. Unter ihnen ist die Subpopulation der Matrix Hersteller verantwortlich zu produzieren und sezernieren die extrazelluläre Matrix des Biofilms wesentlichen für die Biofilmbildung 11,19. Daher ist die Differenzierung von Matrix-Produzenten ein Markenzeichen der Biofilmbildung bei B. subtilis. Wir haben fluoreszierenden Reporter zur Visualisierung und Quantifizierung der Subpopulation der Matrix Produzenten in Biofilmen von B. subtilis 15,19,22-24. Konkret haben wir beobachtet, dass die Subpopulation der Matrix Hersteller in Reaktion auf das Vorhandensein von selbst produzierten extrazelluläres Signal Surfactin 25 unterscheidet. Interessanterweise wird Surfactin von einer Subpopulation von spezialisierten Zellen unterscheidet sich von der Subpopulation von Matrix 15 Herstellern produziert. Wir haben in diesem Bericht die technischen Ansatz notwendig, visualisieren und quantifizieren die Subpopulation von Matrix-Produzenten und Surfactin Produzenten innerhalb der Biofilme von B. subtilis beschrieben. Um dies zu tun, sind fluoreszierende Reporter von Genen für Matrix-Produktion und die Produktion benötigten Surfactin in das Chromosom von B. eingefügt subtilis. Reporter werden nur in einer Subpopulation von spezialisierten Zellen exprimiert. Dann können die Subpopulationenüberwacht mittels Fluoreszenzmikroskopie und Durchflusszytometrie (siehe Abb. 1). Die Tatsache, dass unterschiedliche Subpopulationen von spezialisierten Zellen in vielzelligen Gemeinschaften von Bakterien koexistieren gibt uns eine andere Perspektive über die Regulation der Genexpression in Prokaryoten. Dieses Protokoll befasst sich mit diesem Phänomen experimentell und es kann leicht zu einer anderen Arbeits-Modell angepasst werden, um die molekularen Mechanismen der phänotypischen Heterogenität innerhalb einer mikrobiellen Gemeinschaft aufzuklären.

Watch this Scientific Journal Video about Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry at JoVE.com

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Mikrobielle Biofilme sind in der Regel bestehend aus Subpopulationen von spezialisierten Zellen. Einzelzell-Analyse dieser Subpopulationen setzen die Verwendung von fluoreszierenden Reporter. Hier beschreiben wir ein Protokoll zu visualisieren und überwachen mehrere subpopulationswithin<em&gt; B. subtilis</em&gt; Biofilmen mittels Fluoreszenzmikroskopie und Durchflusszytometrie.

Einzel-Zell-Analyse Bacillus subtilis Biofilme mittels Fluoreszenzmikroskopie und Durchflusszytometrie

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

Research

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Immunology and Infection

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

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Prävention von Hitzestress Unerwünschte Wirkungen bei Ratten durch<em&gt; Bacillus subtilis</em&gt; Dehnungs

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Methodologien für das Studium<em&gt; B. subtilis</em&gt; Biofilme als Modell für kleine Molekül-Inhibitoren Biofilm Charakterisieren

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

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Fluoreszenz-aktivierte Zellsortierung zur Reinigung von Plasmazytoide dendritischer Zellen aus dem Knochenmark der Maus

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

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Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

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Einzellige Analyse der Immunophenotype und Produktion von Zytokinen im peripheren Blut über Masse Zytometrie

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Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...