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Monitorización de GEMs en el ambiente. Marcadores

Monitorización de GEMs en el ambiente. Marcadores. Why monitor domesticated microbial inoculants in nature?. Risk assessment of GMMs Performance/behaviour studies-Ag/Biotech. applications Biological pesticides Bioremediation Biological fertilizers (Rhizobia)

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Monitorización de GEMs en el ambiente. Marcadores

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  1. Monitorización de GEMs en el ambiente. Marcadores

  2. Why monitor domesticated microbial inoculants in nature? • Risk assessment of GMMs • Performance/behaviour studies-Ag/Biotech. applications • Biological pesticides • Bioremediation • Biological fertilizers (Rhizobia) • Basic studies of microbial ecology

  3. Questions to address: • How many cells are present? • Are the cells alive? • Are the cells metabolically active? • How are the cells distributed? • Can the cells perform their intended tasks? • What effect do the cells have on the natural microbial diversity?

  4. Marker Genes Marker Genes Molecular Probes

  5. Marker genes as specific monitoring tools- I - LacZ protein (b-Galactosidase) Well studied and widely used Activity absent in Pseudomonadaceae Different substrates: X-Gal, ONPG, MUG Background activity Visible only in big amounts of cells (colonies) - XylE protein (Catecol 2-3 dioxigenase) Detection of life cells Impredictability (inactivated by O2…)

  6. - LacZ protein - XylE protein

  7. Marker genes as specific monitoring tools- II - Firefly luciferase (luc) or bacterial luciferase (luxAB) Monitor metabolically active cells in the population Detect light emission - Luminometry - Microscopy + sensitive cameras - GFP (gfp): Enumerate total cell population Regardless of physiological status Detect by fluorescence-based methods - Flow cytometry - Fluorescence microscopy

  8. Bioluminiscencia 1. Origen eucariótico (genes luc luciérnaga) LH2 + ATP + O2 CO2 + oxiluciferina + AMP+ luz Mg2+ luciferasa 2. Origen bacteriano (genes lux Vibrio / Photobacterium) FMNH2 + RCHO + O2 H2O + ROOH + FMN + luz Mg2+ luciferasa

  9. Bioluminiscence luxCDABEAB code for the luciferase CDE code for luciferin biosynthesis Strategies: Introduce the whole operon Constitutively luminescent bacteria ~8kb operon, interference with FA biosynthesis Introduce the luciferase Luciferin has to be externally added Reaction always depends on reducing power -> cell status

  10. Green fluorescent protein (GFP de Aequorea victoria) Fluorescencia verde al excitarse con luz UV o azul- sin sustrato ni cofactor Fluorescencia

  11. gfp/luxAB-tagged bacteria Confocal microscopy Cryosection Nycodenz density gradient Fluorescence stereomicoscopy Bacterial fraction Flow cytometry (gfp-tagged cells) Luminometry (lux-tagged cells)

  12. P. fluorescens SBW25 in soil

  13. Confrontation studies with antagonistic fungal strains Trichoderma harzianum - GFP

  14. Marker Genes: monitorisation of E. Coli-GFP colonisation in whole animals

  15. E.coli-GFP infecting peritoneal cavity

  16. Marker Genes Molecular Probes

  17. Molecular probes to detect GEMs Immunological techniques DNA probes PCR-based methods

  18. Immunological techniques - Fluorescent microscopy (single cells) - ELISA (>100 cells) Advantages: Highest specificity (serotyping) Detection at single-cell stage Drawbacks: Cross-reaction Auto-fluorescence Epitope expression

  19. Rhizobium sp. Bradirhizobium sp.

  20. DNA probes - Taxonomic probes - Phylogenetic probes Advantages: Taxonomic level specificity Sensitivity of 16S probes Direct detection of interesting activities Drawbacks: Specificity > species level Crossreaction (diversity unknown)

  21. 16S RNA

  22. Fluorescence in situ hybridization (FISH)

  23. FISH Taxonomic probes In situ hybridization of a vertical biofilm slice with a NIT3-labeled probe specific for the genus Nitrobacter (red stain cluster) correlated to oxygen and nitrate gradients measured by microelectrodes.

  24. FISH Functional probes 20 µm Confocal microscopic image of a bacterial aggregate thin section after hybridization with a Cy3-labeled probe specific for nitrite-oxidizing Nitrospira sp. (red) and a Cy5-labeled probe specific for ammonia-oxidizing Nitrosospira sp. (blue).

  25. PCR-based methods - PCR --> RFLP Advantages: Highest sensitivity (1 cell/gr.) In situ detection of activity Drawbacks: Inspecificity Contamination Interference of humic substances Alterations due to sample purification

  26. PCR 16S rRNA genes • Eubacterial primers • 5´primer fluorescent Restriction digestion Separation on sequencing gel T-RFLP(Terminal-Restriction Fragment Length Polymorphism) Total soil DNA

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