Insight into microbial world molecular biology research in environmental microbiology

Transcription

1 Insight into microbial world molecular biology research in environmental microbiology Aleksandra Ziembi ska The Silesian University of Technology, Environmental Biotechnology Department MICROBIAL WORLD 1

2 A few reasons why we need to look into microbial world How to name the uncultured ones? Which we can use for biotechnological purposes (e.g. wastewater purification, bioremediation)? Which possess patogenic features and how to distroy them? 2

3 MICROBIAL DIVERSITY possible to estimate? BIOCENOSIS 3

4 uncultivable microbes >95% Why molecular methods? analysis without previous cultivation repeteable fast sensitive 4

5 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. 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 5

6 Housekeeping genes vs functional genes what is the difference? 16S rrna gene-based monitoring of bacterial community AmoA gene-based monitoring of bacterial community Molecular methods Based on nucleic acids hybridization Based on PCR amplification 6

7 Fluorescent in situ hybridisation - FISH Particular sequence searching using fluorescently labelled probe. DNA isolation not necessary. Samples are fixated before the procedure. FISH Observation in fluorecent or confocal microscope and particular sequence searching without DNA isolation. 7

8 PCR useful molecular biology tool for xeroxing genetic material DGGE Denaturing 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. 8

9 DGGE enables sequence identification and phylogenetic research. 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. Community qualitative analysis FISH Estimates the character and location of particular bacteria (e.g. activated sludge nitryfiers); enables quantitative analysis (percentage of particular groups of bacteria). Community quantitative analysis 9

10 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) 10

11 Aleksandra Ziembi ska The Silesian University of Technology, Environmental Biotechnology Department Akademicka Gliwice, Poland 11