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Monitorización de GEMs en el ambiente. Marcadores

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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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Presentation Transcript
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?
slide4

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…)

slide6

- 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

slide8

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

slide9

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

slide10

Green fluorescent protein (GFP de Aequorea victoria)

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

Fluorescencia

slide11

gfp/luxAB-tagged

bacteria

Confocal microscopy

Cryosection

Nycodenz density

gradient

Fluorescence stereomicoscopy

Bacterial fraction

Flow cytometry

(gfp-tagged cells)

Luminometry

(lux-tagged cells)

slide19

Marker Genes

Molecular Probes

slide20

Molecular probes to detect GEMs

Immunological techniques

DNA probes

PCR-based methods

slide21

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

slide22

Rhizobium sp.

Bradirhizobium sp.

slide23

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)

slide27

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.

slide28

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).

slide29

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

slide30

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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