Monitorización de GEMs en el ambiente. Marcadores
This presentation is the property of its rightful owner.
Sponsored Links
1 / 30

Monitorización de GEMs en el ambiente. Marcadores PowerPoint PPT Presentation


  • 75 Views
  • Uploaded on
  • Presentation posted in: General

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)

Download Presentation

Monitorización de GEMs en el ambiente. Marcadores

An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -

Presentation Transcript


Monitorizaci n de gems en el ambiente marcadores

Monitorización de GEMs en el ambiente. Marcadores


Why monitor domesticated microbial inoculants in nature

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


Questions to address

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?


Monitorizaci n de gems en el ambiente marcadores

Marker Genes

Marker Genes

Molecular Probes


Marker genes as specific monitoring tools i

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…)


Monitorizaci n de gems en el ambiente marcadores

- LacZ protein

- XylE protein


Marker genes as specific monitoring tools ii

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


Monitorizaci n de gems en el ambiente marcadores

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


Monitorizaci n de gems en el ambiente marcadores

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


Monitorizaci n de gems en el ambiente marcadores

Green fluorescent protein (GFP de Aequorea victoria)

Fluorescencia verde al excitarse con luz UV o azul- sin sustrato ni cofactor

Fluorescencia


Monitorizaci n de gems en el ambiente marcadores

gfp/luxAB-tagged

bacteria

Confocal microscopy

Cryosection

Nycodenz density

gradient

Fluorescence stereomicoscopy

Bacterial fraction

Flow cytometry

(gfp-tagged cells)

Luminometry

(lux-tagged cells)


P fluorescens sbw25 in soil

P. fluorescens SBW25 in soil


Confrontation studies with antagonistic fungal strains trichoderma harzianum gfp

Confrontation studies with antagonistic fungal strains Trichoderma harzianum - GFP


Monitorizaci n de gems en el ambiente marcadores

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


Monitorizaci n de gems en el ambiente marcadores

E.coli-GFP infecting peritoneal cavity


Monitorizaci n de gems en el ambiente marcadores

Marker Genes

Molecular Probes


Monitorizaci n de gems en el ambiente marcadores

Molecular probes to detect GEMs

Immunological techniques

DNA probes

PCR-based methods


Monitorizaci n de gems en el ambiente marcadores

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


Monitorizaci n de gems en el ambiente marcadores

Rhizobium sp.

Bradirhizobium sp.


Monitorizaci n de gems en el ambiente marcadores

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)


Monitorizaci n de gems en el ambiente marcadores

16S RNA


Fluorescence in situ hybridization fish

Fluorescence in situ hybridization (FISH)


Monitorizaci n de gems en el ambiente marcadores

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.


Monitorizaci n de gems en el ambiente marcadores

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).


Monitorizaci n de gems en el ambiente marcadores

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


Monitorizaci n de gems en el ambiente marcadores

  • 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


  • Login