Transcript 무배양적 기법의 예
DGGE를 이용한 환경 Sample의 미생물 군집구조 분석 경기대학교 생명과학과 김재수 머리말 Full name: - Denaturing Gradient Gel Electrophoresis Definition: - A molecular fingerprinting method that separates polymerase chain reaction (PCR)-generated DNA products Principles: - Generating templates of differing DNA sequence that represent many of the dominant microbial organisms - Separating PCR products based on sequence differences that results in differential denaturing characteristics of the DNA - Differing sequences denature at different denaturant concentrations slow movement diff position of band location - Each band theoretically representing a different bacterial population present in the community 2 머리말 - fingerprint similarity can be assessed to determine microbial structural differences between environments or among treatments - used to investigate broad phylogenies or specific target organisms such as pathogens or xenobiotics degraders 3 Entire Process 4 무배양적 기법의 종류 5 무배양적 기법의 장단점 Method Advantage Disadvantage Plate count - fast in process - inexpensive -not detectable for unculturable microorganisms Solo carbon utilization - fast in process - relatively inexpensive - differentiable between microbial communities - not detectable for organisms not capable of using available carbon sources PLFA/FAME - culture free - direct extraction from samples - Influence by external factors G+C content - detectable for even rare members in a community - to provide community structure and relative abundance - requirement of large DNA quantities - low level of taxon resolution Nucleic acid reassociation and hybridization - able to examine total DNA - able to study in situ - lack of sensitivity - requirement of sequences with high copy number PCR/RFLP/ sequencing - to allow a fine identification of uncultured bacteria - informative for diversity, richness and evenness - time-consuming and cumbersome - very expensive equipment (sequencer) - bias due to PCR work DNA microarray - fast analysis of thousands of genes - comprehensive - flexible to any gene - expensive to perform - unknown significance of RNA - uncertain quality control 6 무배양적 기법의 장단점 - sensitive by sample handling possible more than one stable form of ssDNA detectable for only most abundant species not able to separate amplicons harboring different sequences formation of chimaeric equences differential PCR not efficient separation beyond 500 base pairs to migrate at the same position with two different sequences DGGE & TGGE - analysis of a large number of samples - to provide community structure and relative abundance - informative for the structure of active bacterial populations through RT-PCR - possible for taxonomic identification - easy to perform - less time-consuming ARDRA - able to detect structural changes in microbial community - convenience (no more development) - easy to perform - less time-consuming - choice of suitable restriction enzymes - not always possible to make good resolution for high molecular fragments - formation of chimaeric equences - differential PCR T-RFLP - able to detect only the terminal fragments - easy to perform - less time-consuming - formation of chimaeric equences - differential PCR RISA - to provide a finer taxonomic identification - easy to perform - less time-consuming - not enough information on IGS sequence databank - not always possible to make good resolution for high molecular fragments - formation of chimaeric sequences - differential PCR RAPD - rapid and sensitive for revealing differences between similar complex genomes - easy to perform - less time-consuming - - lack of reproducibility not able to provide phylogenetic information formation of chimaeric sequences differential PCR 7 DGGE 원리 8 DGGE 원리 9 2. 본 론 DGGE 분석 OS PDR PDB PUR PUB UDB OS 100.00 PDR 30.43 100.00 PDB 40.00 41.38 100.00 PUR 30.77 53.85 40.63 100.00 PUB 37.50 44.44 57.14 59.26 100.00 UDB 20.69 36.67 76.92 45.16 51.72 100.00 UUB 44.44 39.13 42.31 50.00 45.83 42.31 UUB 10 100.00 DGGE 분석 19K-6 Uncultured γ-proteobacterium clone CTD47B 20K-5 19K-5; 19K-7; 19K-8; 19K-9; 20K-3; 20K-6; 20K-11 Thiothrix fructosivorans strain Q 19K-2 Environmental clone CC-5 20K-7; 20K-10 γ-Proteobacteria Uncultured Pseudomonas sp. clone SR-DGGE-a I-2 I-3; I-13 Pseudomonas sp. WDL5 I-4 I-12 I-5 20K-4 I-14 Uncultured β-proteobacterium clone UCT N117 19K-4 Uncultured β-proteobacterium clone LTUB09814 β-Proteobacteria Uncultured bacterium clone RB13C5 19K-10 20K-1; 20K-2 Sphaerotilus sp. L19 gene 20K-9 Sphaerotilus sp. IF14 19K-3 Uncultured CFB group bacterium isolate DGGE band GC1 20K-8 CFB group bacterium RW262 CFB I-1 Uncultured flavobacterium sp. clone JG37-AG-16 Sphingobacteria I-6; I-10; I-11 Uncultured Cytophagales bacterium strain KIN30 Flavobacteria I-7 Uncultured Cytophagales clone CRE-PA82 I-8 Uncultured freshwater bacterium LCK-64 Unidentified bacterium, clone Sai4P3-55 I-9 ε-Proteobacteria Uncultured ε-proteobacterium clone 33-PA4B00 19K-1 Uncultured bacterium clone LBF6 11 Materials Plate sandwich materials - 16 x 16 cm glass plate - 16 x 14 cm glass plate - 2 - 1 mm spacers - 2 - plate clamps - Pouring stand - Foam gasket - Well comb - Spacing card 12 Materials Gel solutions - 40% Bis-Acrylamide: (37.5: 1, acrylamide: bisacrylamide) - Deionized Formamide - Urea - 50 X TAE (20 mM Tris-acetate, pH 7.4, 10 mM sodium acetate, 0.5 mM Na-EDTA) - 10% Ammonium Persulfate Solution (APS, 0,1 g in 1 ml of nanopure water. Make fresh when required or aliquot 0.5 ml into 1.5 ml microcentrifuge tubes and freeze until needed) - TEMED (N,N,N',N' -tetramethylenediamine) - Glycerol - Nanopure water Gradient wheel GelStar nucleic acid stain DCODE electrophoresis apparatus Power supply 13 Procedure A. Building the gel assembly 14 Procedure B. Pouring the gel 15 Procedure C. Running the gel Gels attached to the core assembly 16 Procedure D. Staining the gel 17 Procedure 지하수seepage에 형성된 biofilm 2. 유류오염 토양 3. 유류오염근권 토양 4. 중금속오염 토양 5. 침출수오염 지하수 6. 유류오염 지하수 7. 광산폐수오염 지하수 8. Biofilter system 1. 18 2. 본 론 무배양적 기법의 예 (FISH) 19 40 20 60 14:0 14 16 18 23.408 21.513 pA 18.127 16:0 18:2 w 6,9 21.311 21.416 무배양적 기법의 예 (PLFA/FAME) 120 20 21.671 21.97221.838 22.365 22.535 22.804 22.933 23.141 23.308 23.590 23.686 23.892 24.049 24.199 24.321 24.423 24.496 24.603 24.872 25.151 25.289 25.545 25.751 25.975 26.171 26.272 26.361 26.480 26.689 26.913 27.064 27.247 27.359 27.465 27.594 27.895 28.052 28.228 28.490 28.659 28.837 28.941 29.035 29.131 29.609 29.376 29.863 15:0i 17.736 15.418 80 15:0a 15.581 15.677 15.823 15.926 16.146 16.492 16.884 17.003 17.292 17.399 17.622 17.910 18.318 18.569 18.951 19.093 19.192 19.317 19.48319.623 19.764 19.901 20.006 20.076 20.372 20.468 20.768 21.062 21.185 12.990 13.161 13.387 13.618 13.726 13.899 14.131 14.480 14.739 15.009 15.174 2. 본 론 Fatty Acid Microbial Group 15:0i, 17:0i, 15:0a, etc.. Gram positive bacteria cy17:0, cy19:0, 18:1D11c Gram negative bacteria (also cy19:0 gm+) 10 Me18:0, 10 Me17:0, 10 Me16:0 Actinomycetes 18:2w6,9, 18:1w9c Fungi 20:4 w6 Protozoan 16:1 w5 Arbuscular mycorrhizal fungi 18:1w8c Methanotrophs FID1 A, (C:\DOCUME~1\JENN\MYDOCU~1\RESEARCH\GC-FID~1\08120312.D) 18:1 w 9c 18:1 w 7c 100 19:0 cy 16:0 10 Me 18:0 10 Me 22 24 26 28 3020 min 2. 본 론 무배양적 기법의 예 (T-RFLP) 21 2. 본 론 Bacteria Methanogens 혐기성 세균의 계통발생적 분류 Sulfate-reducers Phylogenetic Group Species Euryarchaeotes Methanobacterium thermoautotrophicum1) Methanobrevibacter arboriphilicus1) Methanococcus voltae1) Methanococcus termolithotrophicus1) Methanosarcina barkeri1) Euryarchaeotes Archaeoglobus sp.4) δ-Proteobacteria Desulfoarculus sp.4) Desulfobacter sp.4) Desulfobacterium sp.4) Desulfobotulus sp.4) Desulfobulbus sp.3,4) Desulfocapsa sp.3) Desulfococcus sp3,4) Desulfomicrobium sp.4) Desulfomonile sp.4) Desulfonema sp.4) Desulforhopalus sp.3) Desulfosarcina sp.3,4) Desulfotomaculum sp.4) Desulfovibrio sp.4) Thermodesulfobacte ria Thermodesulfobacterium sp.4) Crenarchaeota Sulfolobus acidocaldarius6) Firmicutes Bacillus sp.5) Clostridium sp.6) Staphylococcus aureus5) β-Proteobacteria Rhodoferax ferrireducens7) Thiobacillus ferrooxidans6) Thiobacillus thiooxidans6) γ-Proteobacteria Acinetobacter calcoaceticus5) Acinetobacter johnsonii5) Pseudomonas fluorescens5) Pseudomonas sp.5,6) Shewanella putrefaciens5,6) δ-Proteobacteria Desulfuromonas acetoxidans5) Desulfovibrio sp.6) Desulfovibrio vulgaris6) Geobacter metallireducans6,7) Geobacter sp.2,6) Fe(III)-reducers 22 2. 본 론 혐기성 세균의 계통발생적 분류 Mn(IV)-reducers Firmicutes Bacillus sp.5) Staphylococcus aureus5) γ-Proteobacteria Acinetobacter calcoaceticus5) Acinetobacter johnsonii5) Pseudomonas fluorescens5) Pseudomonas sp.5,6) Shewanella putrefaciens5,6) δ-Proteobacteria Desulfuromonas acetoxidans5,6) Geobacter metallireducans5,6) Geobacter sp.2) Actinobacteria Corynebacterium diphtheriae8) Corynebacterium efficiens8) Corynebacterium glutamicum8) Mycobacterium bovis7) Mycobacterium tuberculosis8) Rubrobacter xylanophilus8) Streptomyces coelicolor8) Crenarchaeotes Aeropyrum pernix8) Pyrogaculum aerophilum8) Euryarchaeotes Haloarcula marismoritui8) Firmicutes Bacillus anthracis8) Bacillus cereus8) Bacillus licheniformis8) Bacillus stearothermophilus8) Bacillus subtilis8) Geobacillus stearothermophilus8) Lactobacillus plantarum8) Selenomonas ruminantium8) Staphylococcus aureus8) Staphylococcus carnosus8) Staphylococcus epidermidis8) Nitrate-reducers 23 2. 본 론 ⍺-Proteobacteria Azospirillum brasilense8) Bradyrhizoium japonicum8) Brucella suis8) Brucella melitensis8) Magnetospirillum magnetotacticum8) Paracoccus denitrificans8) Rhodobacter sphaeroides8) Sinorhizobium meliloti8) β-Proteobacteria Azoarcus sp.2) Bordetella bronchiseptica8) Bordetella parapertussis8) Burkholderia cepacia8) Burkhoderia fungorum8) Burkholderia pseudomallei8) Chromobacterium violaceum8) Ralstonia metallisurans8) Ralstonia solanacearum8) Wautersia eutropha8) γ-Proteobacteria Actinobacillus actinocydetemcomitans8) Escherichia coli8) Haemophilus ducreyi8) Haemophilus influenzae8) Halomonas halodenitrificans8) Pasteurella multocida8) Photobacterium profundum8) Pseudomonas aeruginosa8) Pseudomonas fluorescens8) Psychrobacter sp.8) Salmonella enterica8) Salmonella typhi8) Salmonella typhimurium8) Shewanella oneidensis8) Shigella flexneri8) δ-Proteobacteria Geobacter metallireducans8) ε-Proteobacteria Campylobacter jejuni8) Helicobacter hepticus8) Sulfurospirillum barnesii8) Wolinella succinogenes8) Thermus Thermus thermophilus8) 혐기성 세균의 계통발생적 분류 Nitrate-reducers 24 3. 결 론 오염 지하수 미생물의 군집 특성 Contaminant Reducer Hydrocarbons BTEX Fe(III)-reducer Sulfate-reducer CH4-producer Denitrifier MTBE Aerobe Denitrifier Phylogenetic Group β-Proteobacteria γ-Proteobacteria CFB* β-Proteobacteria Genus Others Pseudomonas Microbacterium Azoarcus Mycobacterium Bradyrhizobium -Benzene은 황환원균 보다 denitrifier에 잘 분해됨 Methylibium - BTEX와 MTBE는 서 로 저해/무해 또는 군 집구조 변화/무변화 Dehalococcoides Chlorinated Compounds (TCE, PCE, PCB) *CFB: Fe(III)-reducer Sulfate-reducer Denitrifier -Proteobacteria Low G+C GramFirmicutes GNS bacteria Desulfitobacterium Dehalobacter Geobacter Pelobacter Desulfurromonas Clostridium (PCB) Pseudomonas (carbon tetrachloride) -Dehalococcoides ethnogenes 는 PCE 부터 ethene까지 reducing Cytophaga-Flexibacter-Bacteridetes 25 2. 본 론 무배양적 기법의 예 (RISA) 26 2. 본 론 무배양적 기법의 예 (RISA) TABLE 1 Frequencies and phylogenetic affiliations of clones derived from the 350-bp band (RISA band 1) Phylotype No. (%) of clones Accession no. of RISA band sequences Phylogenetic affiliation Closest relative % similarity Accession no. of closest relative 1 41 (54) AF268436, AF268437 AF268438, AF268439 Gram-positive low G+C Clostridium tyrobutiricum 85 L08062 2 4 (5.2) AF268434, AF268435 Gram-positive low G+C Clostridium tyrobutiricum 85 L08062 3 2 (2.6) AF268440 Gram-positive low G+C Clostridium tyrobutiricum 85 L08062 RISA band 27