Download Ziembinska

19.7.2010
Insight into microbial
world – molecular biology
research in environmental
microbiology
Aleksandra Ziembi ska
The Silesian University of Technology,
Environmental Biotechnology Department
[email protected]
MICROBIAL WORLD
Ziembi ska A., SUT, Insight into microbial world… Mikkeli, Finland, July, 2010
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19.7.2010
Ziembi ska A., SUT, Insight into microbial world… Mikkeli, Finland, July, 2010
A few reasons why we need to look into microbial
world…
Which we can use for
biotechnological
purposes (e.g. wastewater
purification, bioremediation)?
How to name
the uncultured ones?
Which possess patogenic
features and how to
distroy them?
Ziembi ska A., SUT, Insight into microbial world… Mikkeli, Finland, July, 2010
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19.7.2010
MICROBIAL DIVERSITY –
possible to estimate?
Ziembi ska A., SUT, Insight into microbial world… Mikkeli, Finland, July, 2010
BIOCENOSIS
Ziembi ska A., SUT, Insight into microbial world… Mikkeli, Finland, July, 2010
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19.7.2010
uncultivable microbes
>95%
Ziembi ska A., SUT, Insight into microbial world… Mikkeli, Finland, July, 2010
Why molecular
methods?
analysis without
previous cultivation
repeteable
fast
sensitive
Ziembi ska A., SUT, Insight into microbial world… Mikkeli, Finland, July, 2010
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19.7.2010
Universal bacterial molecular marker - what is that?
Particle present in every bacterial cell in
abundance;
large molecule;
functionally stable;
if it is possible, it should belong to the
housekeeping genes;
Possess both, evolutionary
conservative and changeable
fragments, that enables
identification and
evolutionary relationships research.
Ziembi ska A., SUT, Insight into microbial world… Mikkeli, Finland, July, 2010
Why 16S rRNA gene is universal?
protein translation: universal
no horizontal transfer
convenient length : 1500 bp
highly conserved regions as well
as species-specific regions
large databases
16S rRNA
ribosome
Ziembi ska A., SUT, Insight into microbial world… Mikkeli, Finland, July, 2010
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19.7.2010
Housekeeping genes vs functional genes
– what is the difference?
AmoA gene-based
16S rRNA gene-based monitoring of
monitoring of bacterial
bacterial community
community
Ziembi ska A., SUT, Insight into microbial world… Mikkeli, Finland, July, 2010
Molecular methods
Based on nucleic acids
hybridization
Based on PCR amplification
Ziembi ska A., SUT, Insight into microbial world… Mikkeli, Finland, July, 2010
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19.7.2010
Fluorescent in situ hybridisation - FISH
Particular sequence searching using fluorescently labelled probe. DNA
isolation not necessary. Samples are fixated before the procedure.
Ziembi ska A., SUT, Insight into microbial world… Mikkeli, Finland, July, 2010
FISH
Observation in
fluorecent or
confocal microscope
and particular
sequence searching
without DNA
isolation.
Ziembi ska A., SUT, Insight into microbial world… Mikkeli, Finland, July, 2010
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19.7.2010
PCR – useful molecular biology tool
for „xeroxing” genetic material
Ziembi ska A., SUT, Insight into microbial world… Mikkeli, Finland, July, 2010
DGGE – Den
Denaaturing Gradient Gel Electrophoresis
DGGE is based on
PCR products
separation due to
its sequence, not
DNA fragment size.
DNA isolation
before the
procedure is
necessary.
It enables fingerprints obtainment, useful in different
samples comparison. Cutting DNA band off the gel in order
to sequence and identify it is also possible.
Ziembi ska A., SUT, Insight into microbial world… Mikkeli, Finland, July, 2010
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19.7.2010
DGGE enables sequence identification
and phylogenetic research.
Ziembi ska A., SUT, Insight into microbial world… Mikkeli, Finland, July, 2010
Combination of DGGE and FISH - complementary results
DGGE
Helps in changeability
and biodiversity
research. Samples
can be compared
among the group and
the dominants can be
identify by
sequencing.
FISH
Estimates the
character and location
of particular bacteria
(e.g. activated sludge
nitryfiers); enables
quantitative analysis
(percentage of
particular groups of
bacteria).
Community qualitative
analysis
Community quantitative
analysis
Ziembi ska A., SUT, Insight into microbial world… Mikkeli, Finland, July, 2010
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19.7.2010
Restriction enzymes – molecular scissors
for bacterial diversity analysis
Restriction enzymes based methods examples:
RFLP (ang. Restriction Fragment Length
Polymorphism)
- ARDRA (ang. Amplified Ribosomal DNA
Restriction Analysis)
- AFDRA (ang. Amplified Functional DNA
Restriction Analysis)
T-RFLP (ang. Terminal
Restriction Fragment Length
Polymorphism)
Ziembi ska A., SUT, Insight into microbial world… Mikkeli, Finland, July, 2010
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19.7.2010
Aleksandra Ziembi ska
The Silesian University of Technology,
Environmental Biotechnology Department
Akademicka 2.
44--100 Gliwice, Poland
44
E-mail: [email protected]
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