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Pooja Sethiya Sushma Patil
Name of the Guide
. The gut microbiota was recently proposed as an environmental factor responsible for the control of body weight and energy metabolism. Metabolic regulation and bacterial diversity of the gut plays important role in defining health makeup of an individual. which is closely linked to obesity and metabolic disorders such as type 2 diabetes.INTRODUCTION Human adult harbors ~10 times more microbial cells than its own.
Functions of Microbiota .
various techniques such as 16S rRNA gene sequencing are used.INTRODUCTION Microbiota has been studied by culture dependent techniques. . Studies suggest that 60% to 80% of the observable bacteria cannot be cultured. Hence.
The microbiota of Indian population will be different as Indian population differs in– – – – dietary habits. exposure to indoor .Rationale of the study Microbiota effectively contributes to various metabolic as well as immunological processes as reported within the western population. Lifestyle degree of sanitation . However. outdoor air pollution. data is not available for Indian population.
Aims and objectives To study gut microbial diversity within Indian individuals. To investigate microbiota in diabetic individual which can be used for future research. . Compare inter-individual microbiota.
Protocol • Stool Sample Collection • Isolation of bacterial community DNA • PCR with 16S rRNA primers • Purification of PCR products • Cloning of purified product I II III IV V VI VII • Patching of clones and sequencing • Bioinformatic analysis .
Purification of PCR Product Stool Samples Total genomic DNA isolation PCR with universal 16S rRNA gene primers and PCR product Purification 1Kb 1kb Ligation of PCR product in TOPO 4 vector SM51 SF51 SM88 .
Transformation into E. coli DH5a Screening of colonies for inserts by Colony PCR Purification of PCR products Sequencing using BigDyeTM ABI techology Sequence Processing by Bioinformatics code Involves high-end computing Sequence based homology search in NCBI databaseBLAST tool .
Results and Discussions 50 45 40 35 NO OF OTU”S 30 25 20 15 10 5 0 0 20 40 60 80 NO OF CLONES 100 120 140 160 SM51 SM88 SF51 .
Firmicutes .Abundance of OTUs at Family Level – Sample SM51 unclassified_Clostridiales 16% Lactobacillaceae 6% Veillonellaceae 24% Lachnospiraceae 43% Ruminococcaceae 11% Phylum .
99 GQ898852 uncultured bacterium.w2k SM51 C94 SM51 C167 GQ896923 uncultured bacterium. 29A-f1 100 SM51 C9 SM51 C95 DQ905899 uncultured bacterium. SJTU_B_07_34 100 EF403212 uncultured bacterium. S3-162 SM51 C62 SM51 C33 100 AB506300 uncultured_bacterium_R-C-B06 100 100 AB506301 uncultured bacterium. SJTU_C_03_05 100 AM275473 uncultured bacterium.SM51-Diabetic 33 23 Family . 014C-E4 EF403972 uncultured bacterium.78 SM51 C104 100 GQ898614 uncultured bacterium. R-C-B07 30 96 83 94 92 89 Family .Lachnospiraceae 37 100 77 100 100 Unclassified Clostridiales GQ897302 uncultured bacterium C4-188 GQ159255 uncultured bacterium16slp98-1b12.Veillonellaceae 0. C2-200 100 EF403389 uncultured bacterium. SJTU_A1_6_8 SM51 C2 SM51 C153 100 DQ905704 55 100 EF404181 uncultured bacterium. AP07K. . SJTU_C_05_85 100 DQ90575 uncultured bacterium.01 Fig: Neighbour-joining tree (1000 boostraps) was generated using 16S rRNA gene fragement. 29c-d3 98 100 GQ898790 uncultured bacterium. SJTU_A1_7_60 100 94 EF402393 uncultured bacterium. S4-146 SM51 C8 DQ905668 uncultured bacterium. 29A-f1.
Phylum level abundance of OTUs – Sample SF51 Bacteroidetes 2% Proteobacteria 21% Firmicutes 77% .
7 SF 51-NON DIABETIC Class . Okuchi-16 82 EU163449 Streptococcus gallolyticus subsp. p-4496-6Wb3 GQ159307 uncultured bacterium. M2-9 75 EF403681 uncultured bacterium. Okuchi-2 67 EU163440 Streptococcus gallolyticus subsp. SJTU_E_08_57 99 SF51 C19 73 EF401943 uncultured bacterium.w2k 48 SF51 C62 SF51 C14 SF51 C169 100 EU530370 uncultured Catenibacterium sp. CFT19F10 SF51 C161 SF51 C87 SF51 C42 85 EU530262 uncultured Faecalibacterium sp. SJTU_E_08_31 100 EF400076 uncultured bacterium. SJTU_B_01_63 76 EF401950 uncultured bacterium. 16slp116-5c05. gallolyticus. SJTU_A2_05_32 89 99 EF400055 uncultured bacterium. gallolyticus.p1k 100 GQ158077 uncultured bacterium. 16slp116-1a11. 16slp94-2e11. M4-82 100 65 EU071505 uncultured Catenibacterium sp. EHFS1_S11a SF51 C165 SF51 C20 SF51 C88 SF51 C127 SF51 C69 SF51 C164 95 AB303624 Streptococcus gallolyticus subsp.. SJTU_B_01_73 SF51 C3 SF51 C178 SF51 C113 100 100 2 25 13 73 47 93 77 88 41 62 26 60 Class .. SJTU_G_02_82 100 EF405182 uncultured bacterium.p1k SF51 C130 99 EF405367 uncultured bacterium. pasteurianus 100 AB303623 Streptococcus gallolyticus subsp.Gammaproteobacteria .. pasteurianus EU124835 rumen bacterium R3_91_A SF51 C76 DQ455879 uncultured bacterium. SJTU_G_06_91 86 SF51 C92 GQ158076 uncultured bacterium.Clostridia AF371680 uncultured bacterium.
Phylum level abundance of OTUs – Sample SM88 Proteobacteria 8% Firmicutes 92% .
A2-51 AF357566 bacterium mpn-isolate group 18 95 89 59 AY975518 uncultured bacterium. B12 100 90 . 31g07 99 DQ824482 uncultured bacterium.SM88 NON DIABETIC EF399720 uncultured bacterium. 7-11 91 FN667451 uncultured compost bacterium. 29c-g4 69 DQ793264 uncultured bacterium.02 78 100 Fig: Neighbour-joining tree (1000 boostraps) was generated using 16S rRNA gene fragement. PS3297 93 AF371696 uncultured bacterium. . p-3424-SwA2 72 AY952453 Halomonas sp. SJTU_G_07_36 GQ897609 uncultured bacterium. SJTU_C_03_33 AB034089 uncultured rumen bacterium 4C3d-19 15 100 50 AF371499 uncultured bacterium.w2k 175 79 51 U95028 Megasphaera elsdenii. SJTU_E_02_56 EF404581 uncultured bacterium. AB305243 Halomonas sp. oral clone VeillE4 66 GQ157131 uncultured bacterium. p-1934-s962-3 83 21 16 17 AB034085 uncultured rumen bacterium 3C3d-7 0. SJTU_C_13_08 56 167 172 90 100 EF405054 uncultured bacterium. SJTU_C_01_11 90 99 128 EF403995 uncultured bacterium. S2 AY196919 Megasphaera elsdenii. RL199_aaj41e03 92 129 102 100 160 EF515668 uncultured bacterium. RL304_aal76e09 99 AY995765 Veillonella sp. 16slp101-3g03. CA3-3XX DQ905918 uncultured bacterium. N980 EF403829 uncultured bacterium.
16 14 12 10 8 6 4 2 SF51 SM51 SM88 0 .Comparison of relative abundance of OTUs in Phylum – Firmicutes within and between families.
Number of Firmicutes were more in case of diabetic patients than the non-diabetic individual. .Conclusions Inter-individual variation of gut bacterial communities were observed.
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