1 1 2 CADLIVE E-CELL
3 GEM CE/MS MGF WT MGF 4 2
WT E-Cell MASK 5 CE-MS, CE- TOFMS LC-MS 24, 5 9 2 0 6 3
CE-MS CE MS Soga, T. et al. (2002) Anal Chem. 74, 2233-2239 7 CE-MS 859 452, 269, 86 8 4
LC-MS 9 CE-TOFMS CE-MS/MSMS/MS CE-MS 10 5
SGP 67 WB 26 2D-DIGE 11 13 C GC-MSNMR CE-MS 12 6
Real time RT-PCR 85 rrna 2 ) 8 DNA 13 Escherichia coli BW25113 (WT) or Keio : 0.2 [hr -1 ] 2 = 3.5 [hr] 37 ph7.0 CE-TOFMS 578 67 26 2D-DIGE GC-MS, NMR CE-MS Real time RT-PCR 95 DNA 14 7
E. coli K-12 15 0.1, 0.2, 0.4, 0.5, 0.7 [hr -1 ] Keio 0.2 [hr -1 ] galm,glk,pgi,pfka,pfkb,fbp,fbab,gapc,gpma,gpmb, pyka,pykf,ppsa,pgm zwf,pgl,gnd,rpe,rpia,rpib,tkta,tktb,tala,talb / 16 8
17 (Shotgun Proteomics) 18 9
19 (PCR) rpiarpib 20 10
11 21 22 S K P K K P S E k v P m S m eq t cat + + =,, 1 CE/MS in vitro or Flux or Michaelis-Menten
E-Cell 3 0.1, 0.2, 0.5 [hr -1 ] 0.4[hr -1 ] 23 zwf Zwf (G6PDH) = 0 0 0 R5P 24 12
pgi WT / Acetyl CoA KO Case 1 KO Case 2 25 rpe WT / Case 1 Case 2 KO KO 26 13
pfka PfkA (Km for F6P) G6P 27 Chassagnole(2002) G6P F6P FDP GAP PEP PYR 6PG D = 0.1 [hr -1 ] 3 XTime [sec] YNormalized concentration [-] PYRactivate 28 14
Hoque et al. (2005) PGactivate XTime [sec] YNormalized concentration [-] 29 Crabtree effect NH 4 P i PTS kcat 30 15
HDS MASK 50% DNA 31 Escherichia coli K12 MG1665 Metabolites 940 Enzymes 1178 Reactions 1179 Genome Sequence attgaacgct tgcttcgctg gataactact caccaaggcg GEM SYSTEM Set of Reactions = Metabolic Pathways SWISS-PROT KEGG The GEM system automatically builds static cell models from genomic sequences using various databases. Arakawa, K. et al (2006) BMC Bioinformatics 7, 168 32 16
/ Dynamic model X 2 v 1 X 4 X n X 1 X 3 X 12 X 11 v 9 v 8 Stoichiometric model v 2 X 5 v 3 X 6 X 10 X 8 v 7 X 9 v 6 v 4 X 7 v 5 dx 5 /dt = V 2 + V 3 dx 11 /dt = V 8 + V 9 Yugi, K. et al. (2005) Theor. Biol. Med. Model. 2, 42 33 MASK Delay 1 min 6 A a R R x x =2 A =3R x x Regulated gene R B =R 2 x Regulator gene 4 B b Delay 5 min l Kb [ RNAP ] i Vsyn = Ka Kc 1 + Kb [ RNAP ] j m = 0 [ A ] h [ R ] = 0 i g j R Yugi, Y. et al (2005) BMC Bioinformatics 6, 299 S. Cerevisiae (Spellman et al. (1998) Mol. Biol. Cell 9, 3273-3297) 34 17
18 35 MOMA FBA ECF ECF 36 MOMA FBA ECF 4 CADLIVE CADLIVE
37 Nucleoc acods Res. 2003 Bioinformatics 2005 Genome informatics, 2004 38 19
σ 32 mrna FF sensor FB sensor Computer σ 32 σ 32 DnaK Heat Shock Plant PNAS, 2005 RNAP Actuator RNAP σ 32 σ 32 DnaK FtsH σ 32 FtsH DnaK HSP(DnaK, FtsH, Lon) genes () Genome Res. 2005 39 40 20
WTW3110 MGF-01 MGF-01 0 41 MGF MGFMGF-01 W3110 42 21
CE-TOFMS WT (W3110) MGF-01 43 44 22
23 45 S/N<3, Area<1000, 46 CE-MS Glycolate Trp Tyr Phe PRPP His Glc G6P F6P F1,6BP DHAP G3P PEP Pyruvate AcCOA OAA Malate Fumarate Succinate SucCoA akg IsoCit Citrate GOX GLcn6P Ribo5P Ribu5P 1,3BPG SH7P F6P X5P GA3P E4P GA3P Glconate Glu Gln Arg Pro Lac Asp Asn Thr Ser Gly Cys Met Ile Val Leu Lys G2P Ala AcOH Glycerophosphate Glycogen G1P 2,3BPG cis-aco camp AMP ADP ATP NAD NADH NADP NADPH PA PS CMP-DG CMP 2,3BPG
MGF MGF,,, W3110 MGF 47 CE-MS 6 4 2 48 24
Human Metabolome Technologies Inc. Since 2003 Keio invested HMT 2003.11.27 49 50 25
26 51 52 Control (Cy5) NaCl (Cy3) (Cy5+Cy3) 5 10 15 20 30 25 97 66 45 30 20 14 (kda) pi 3 10
1. 2. 2-1 2-2 SOM SOMAICDNA SOM 3. 3-1 GPD1 3-2 NaClENA1CUP1 3-3 TRP1TRP5 53 54 27