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1 Gene Essentialities of Bacterial Systems TOMOYA BABA Research Organization of Information and Systems Transdisciplinary Research Integration Center National.

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Presentation on theme: "1 Gene Essentialities of Bacterial Systems TOMOYA BABA Research Organization of Information and Systems Transdisciplinary Research Integration Center National."— Presentation transcript:

1 1 Gene Essentialities of Bacterial Systems TOMOYA BABA Research Organization of Information and Systems Transdisciplinary Research Integration Center National Institute of Genetics, Misima, Japan Joint work with: Barry L. Wanner (Purdue Univ., US) Masaru Tomita (Keio Univ., Japan) Hirotada Mori (NAIST, Japan) ISSSB 11, Nov. 16th 2011, Shonan Village Centre, Japan Introduction of following two talks: Takehide Soh Predicting Gene Knockout Effects by Minimal Pathway Enumeration Takeyuki Tamura Predicting essential genes via impact degree on metabolic networks

2 2 Genes (Genome) Proteins Robot Blueprint Parts Systems Engineering Organism Human E. coli Systems Biology

3 3 Escherichia coli K-12 Genome sequenced at 1997 Revised at 2006 Re-annotation at 2006 Well-characterized Bacteria Science (1997) Riley M., Nuc. Acid. Res., 34, 1-9 (2006) 54% 32% 14%

4 4 Ultimate Goal : Comprehensive Understanding of Life ORF mRNA Transcription E. coli cell Translation Metabolites Chromosome Genome DNA Regulation Replication Cell-Division Transport Secretion Signal-Transduction etc Enzyme Promoter Unknown Yes it is, however, life is so complicated systems. Simple Question Which genes are essential for the life systems? Protein 1,

5 5 Gene-Knockout Experimental Method Red recombinase Chromosome Target Gene Step 2. Transformation pKD46 (ts-ori) kan Step 1. PCR of Maker gene E. coli Wild type cell (BW25113) (30 ) bla 37, LB(Km + ) Chromosome Gene-knockout cell 1, Precise design 2, Complete deletion Datsenko K.A., Wanner B.L., (2000) Proc. Natl. Acad. Sci. 97, 6640.

6 6 P1 P2 H1 H2 Kanamycin resistance gene FRTFRT 21 bp geneB (target) geneAgeneC H1H2 Met Step 1. PCR of Kanamycin resistance (kan) gene Designing for In-frame Deletion FRT geneA geneC FRT 21 bp 34 a.a. (102 bp) MIPGIRRPAVRSSTSLGSIGTSKQLQPT+6aa ( Step 3. Elimination of kan gene) FLP recombinase FRTFRT Kanamycin resistance gene Step 2. Transformation Red recombinase SD of downstream gene

7 7 Total targeted ORFs Keio collection Essential 3912 with annotation 73 Small ORFs (no longer annotated) 303 Single Gene-Knockout Results C: 1655 E: 100C: 331 E: 1819 Named genes 2157 y genes 1755 ORF evidence E; experimental C; computational Baba T., (2006) Mol. Syst. Biol. 2006, 2, 8. Result 303 genes (7% of total genes) are essential in E. coli K-12.

8 8 Classification of E. coli K-12 Gene Functions % of Essential or Total ORFs Essential 303 COGs (Clusters of Orthologous Groups of proteins) category Information storage and processing ( 107 / 695 ) JTranslation, ribosomal structure and biogenesis KTranscription LDNA replication, recombination and repair Cellular processes ( 85 / 977 ) DCell division and chromosome partitioning OPosttranslational modification, protein turnover, chaperones MCell envelope biogenesis, outer membrane NCell motility and secretion PInorganic ion transport and metabolism TSignal transduction mechanisms Metabolism ( 90 / 1,444) CEnergy production and conversion GCarbohydrate transport and metabolism EAmino acid transport and metabolism FNucleotide transport and metabolism HCoenzyme metabolism ILipid metabolism QSecondary metabolites biosynthesis, transport and catabolism Poorly characterized ( 59 / 2,109) RGeneral function prediction only S, U or VFunction unknown No COG assignment Total Baba T., (2006) Mol. Syst. Biol. 2006, 2, 8. Result 2.90 genes (6% of metabolic genes) are essential.

9 9 Growth of Gene-Knockout Mutants LB (22 hrs) OD 600 AVE-2SD MOPS-0.4% Glucose (48 hrs) OD 600 AVE+2SD AVE-2SD AVE+2SD COGs category ; Information storage and processing ; Cellular processes ; Metabolism ; Poorly characterized ; No COG assignment Amino Acid 81 Genes Auxotroph Conditionally Essential (Metabolism Genes) Nucleotide 23 Genes Baba T., (2006) Mol. Syst. Biol. 2006, 2, 8. Result genes (7% of metabolic genes) are conditionally essential in glucose minimum medium.

10 10 Metabolic Pathway of E. coli (1,163 genes / 1,119 metabolites) Glucose TCA Glycolysis Pentose phosphate pathway (Detour pathway) Pyruvate Result 4.A lot of detour pathways are existing in E. coli metabolic pathway.

11 11 Glucose Minimal Medium GLC G6PFBP ATP ADP GAP DHAP NAD NADH BPG ADP ATP 3PG2PGPEPPYR 6PGACoA FORM LAC F6P OAA pfkA pfkB fbaA fbaB pgi tpiA gpmA gpmB ppsA ATP ADP gapA gapC pgk enofbp G1P pgmATP ADP glk GLC galM pykA pykF E4P S7P X5P E4P R5P, S7P 4 Essential Genes (Far from Detour pathway?) Pentose phosphate pathway (Detour pathway) TCA Cell Growth of Glycolysis Gene Knockouts in E. coli Glucose Time (h)

12 12 Intracellular Expression of E. coli Cells mRNA Proteins Metabolites rpe Ishii N., Science, 316, 593 (2007) Metabolites 134 Proteins 68 mRNA 85 Gene-Knockouts Glycolysis Pentose phosphate pathway (Detour pathway)

13 13 Robustness of Metabolic Network Metabolites Proteins Metabolic Flux (path-flow) Forward (Wild type) Reverse Keeping Metabolic Balance Regulation of Gene Expression Pentose phosphate pathway (Detour pathway) Ishii N., Science, 316, 593 (2007) Result 5. E. coli cells regulate the gene expression level for keeping metabolic balance.

14 14 Conclusions genes (7% of total genes) are essential in E. coli K genes (6% of metabolic genes) are essential genes (7% of metabolic genes) are conditionally essential in glucose minimum medium. 4.A lot of detour pathways are existing in E. coli metabolic pathway. 5. E. coli cells regulate the gene expression level for keeping metabolic balance. Further Question What is meaning of metabolic essential and conditional essential genes in E. coli? Are the detour pathway lengths (steps) related for them? Takehide Soh Predicting Gene Knockout Effects by Minimal Pathway Enumeration Are there another type or meaning of metabolic essential genes in E. coli? Takeyuki Tamura Predicting essential genes via impact degree on metabolic networks


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