#METABOLOMICS WORKBENCH pwangikar_20181018_234358 DATATRACK_ID:1547 STUDY_ID:ST001080 ANALYSIS_ID:AN001763 PROJECT_ID:PR000723 VERSION 1 CREATED_ON October 26, 2018, 7:12 pm #PROJECT PR:PROJECT_TITLE 13C Labeling Analysis of Synechococcus sp. PCC 7002 PR:PROJECT_TYPE 13C tracing in intracellular metabolites of PCC 7002 PR:PROJECT_SUMMARY An experiment conducted for Metabolic Flux Analysis of wild-type cyanobacterium PR:PROJECT_SUMMARY Synechococcus sp. PCC 7002 PR:INSTITUTE Indian Institute of Technology Bombay (IIT Bombay) PR:DEPARTMENT Department of Chemical Engineering PR:LABORATORY Bio systems Engineering Lab PR:LAST_NAME Wangikar PR:FIRST_NAME Pramod PR:ADDRESS IIT Bombay PR:EMAIL wangikar@iitb.ac.in PR:PHONE 2225767232 PR:FUNDING_SOURCE Department of Biotechnology (DBT), PAN-IIT Centre for Bioenergy #STUDY ST:STUDY_TITLE Dynamic labeling of intracellular metabolites in PCC 7002 ST:STUDY_TYPE Timecourse experiment ST:STUDY_SUMMARY The experiment was conducted in a reactor at a metabolic steady state (OD 730 nm ST:STUDY_SUMMARY = 0.8). 13C labeled sodium bicarbonate was used as a tracer. Tracer ST:STUDY_SUMMARY concentration was 1 g/L. After the introduction of the tracer, the samples were ST:STUDY_SUMMARY collected at time point : 0, 60, 120, 180 and 240 secs. Samples were quenched ST:STUDY_SUMMARY with methanol and extracted using methanol-chloroform-water method. Extracts ST:STUDY_SUMMARY were stored at -80 degree C till LCMS analysis. ST:INSTITUTE Indian Institute of Technology, Bombay ST:DEPARTMENT Department of Chemical Engineering ST:LABORATORY Bio systems Engineering Lab ST:LAST_NAME Wangikar ST:FIRST_NAME Pramod ST:ADDRESS Powai ST:EMAIL wangikar@iit.ac.in ST:PHONE 2225767232 #SUBJECT SU:SUBJECT_TYPE Bacteria SU:SUBJECT_SPECIES Synechococcus sp. PCC 7002 SU:TAXONOMY_ID 1131 SU:GENOTYPE_STRAIN Synechococcus #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - PCC 7002 0 SEC REP 1 Genotype:Wild type SUBJECT_SAMPLE_FACTORS - PCC 7002 0 SEC REP 2 Genotype:Wild type SUBJECT_SAMPLE_FACTORS - PCC 7002 60 SEC REP 1 Genotype:Wild type SUBJECT_SAMPLE_FACTORS - PCC 7002 60 SEC REP 2 Genotype:Wild type SUBJECT_SAMPLE_FACTORS - PCC 7002 120 SEC REP 1 Genotype:Wild type SUBJECT_SAMPLE_FACTORS - PCC 7002 120 SEC REP 2 Genotype:Wild type SUBJECT_SAMPLE_FACTORS - PCC 7002 180 SEC REP 1 Genotype:Wild type SUBJECT_SAMPLE_FACTORS - PCC 7002 180 SEC REP 2 Genotype:Wild type SUBJECT_SAMPLE_FACTORS - PCC 7002 240 SEC REP 1 Genotype:Wild type SUBJECT_SAMPLE_FACTORS - PCC 7002 240 SEC REP 2 Genotype:Wild type #COLLECTION CO:COLLECTION_SUMMARY The experiment was conducted in a reactor at a metabolic steady state (OD 730 nm CO:COLLECTION_SUMMARY = 0.8). 13C labeled sodium bicarbonate was used as a tracer. Tracer CO:COLLECTION_SUMMARY concentration was 1 g/L. After the introduction of the tracer, 15 mL of the CO:COLLECTION_SUMMARY culture were collected at time point : 0, 60, 120, 180 and 240 secs. Samples CO:COLLECTION_SUMMARY were quenched with methanol and extracted using methanol-chloroform-water CO:COLLECTION_SUMMARY method. Extracts were stored at -80 degree C till LCMS analysis. LCMS analysis CO:COLLECTION_SUMMARY was done in negative ion mode using information dependent aquisition (IDA) CO:COLLECTION_SUMMARY method. CO:SAMPLE_TYPE Bacterial cells #TREATMENT TR:TREATMENT_SUMMARY The metabolites were extracted using a methanol-chloroform-water method TR:TREATMENT_SUMMARY described in the "Metabolite Extraction Protocol_PCC 7002" file of the TR:TREATMENT_SUMMARY collection data. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The metabolite extracts of different collected samples were reconstituted in 100 SP:SAMPLEPREP_SUMMARY μL of 50/50 methanol-water mixture and filtered before injecting on LC/MS. SP:SAMPLEPREP_SUMMARY Injection volume was 10 μL. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Shimadzu 20AD CH:COLUMN_NAME Phenomenex Synergi Hydro RP (100 x 2mm, 2.5um) CH:FLOW_GRADIENT 0% B (0 min), 0% B (2min), 35% B (8 min), 35% B (10.5 min), 90% B (15.50 min), CH:FLOW_GRADIENT 90% B (20.5 min) and 0% B (22 min) CH:FLOW_RATE 0.3 mL/minute CH:SOLVENT_A 10 mM tributylamine + 15mM acetic acid in water CH:SOLVENT_B 100% Methanol #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME ABI Sciex 5600+ TripleTOF MS:INSTRUMENT_TYPE Triple TOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak Area MS_METABOLITE_DATA_START Samples PCC 7002 0 SEC REP 1 PCC 7002 0 SEC REP 2 PCC 7002 60 SEC REP 1 PCC 7002 60 SEC REP 2 PCC 7002 120 SEC REP 1 PCC 7002 120 SEC REP 2 PCC 7002 180 SEC REP 1 PCC 7002 180 SEC REP 2 PCC 7002 240 SEC REP 1 PCC 7002 240 SEC REP 2 Factors Genotype:Wild type Genotype:Wild type Genotype:Wild type Genotype:Wild type Genotype:Wild type Genotype:Wild type Genotype:Wild type Genotype:Wild type Genotype:Wild type Genotype:Wild type 3-phosphoglyceric acid 1825000 1809914.748 397000 397864.7562 189600 189758.9978 146500 146531.1831 143800 144545.2416 Acetyl coenzyme A 216300 187144.5723 103000 95456.91977 37110 31014.91095 20430 20311.98449 12440 17766.67371 ADP-Glucose 236363.7529 231200 36776.77178 39160 3497.701153 15740 11782.81148 12170 8055.256363 12020 ADP 1052000 615952.645 2283000 1291472.518 1585000 1436972.627 2090000 1344884.102 1908000 1447495.999 Aspartic acid 166843.8049 169799.7716 46379.87345 46394.01629 29962.95559 30920.09425 21953.58061 25995.42161 27922.77487 25512.83817 ATP 5865000 2619389.732 6068000 2597088.33 4210000 4017615.333 4567000 2500348.74 3580000 2521826.67 Citric acid 16280000 27137352.32 22060000 28545016.49 21300000 29048720.45 20530000 28784886.33 32090000 32332715.78 Glucose 6 phosphate 372300 280300 101200 98670 53930 74170 64950 28140 912.3 27410 Glutamic acid 6454000 6120000 5328000 5517000 5847000 5393000 4624000 4460000 5029000 4782000 Leucine 187332.3245 190645.4362 194909.8116 184260.5519 138918.3898 143141.446 112073.1538 120720.6836 127840.8323 119246.7778 Malate 715771.8932 738800 432258.8547 439300 260719.5945 234500 146536.8448 153700 142233.436 158300 nicotinamide adenine dinucleotide 211900 197184.9309 224800 212751.8614 219600 200295.1679 174000 170269.6746 232700 232441.5314 Sedoheptulose 7 phosphate 561900 444300 95910 80200 67200 64530 48110 49620 59100 51200 Succinic acid 789700 800000 493700 498700 349200 358300 349400 354000 349500 373700 Sucrose 3470000 3497112.158 3144000 3251845.298 3246000 3278608.459 2884000 2906881.366 3229000 3258915.414 Tyrosine 666178.3389 645388.2082 448455.8569 439872.226 310685.8726 310750.3772 217046.6913 218686.6716 229142.4485 217703.2177 UDP-Glucose 199027.6441 171900 62667.22165 61770 25495.70979 20050 12722.99954 12060 7464.802252 3018 Uridine monophosphate 3068000 2565363.449 4767000 4423167.365 4537000 4324243.151 4025000 3851815.727 5162000 5089080.562 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Elemental formula KEGG ID retention index (min) 3-phosphoglyceric acid C3H7O7P C00197 12.29 Acetyl coenzyme A C23H38N7O17P3S C00024 15.36 ADP-Glucose C16H25N5O15P2 C00498 12.54 ADP C10H15N5O10P2 C00008 13.76 Aspartic acid C4H7NO4 C00049 5.14 ATP C10H16N5O13P3 C00002 14.93 Citric acid C6H8O7 C00158 13.6 Glucose 6 phosphate C6H13O9P C00668 7.34 Glutamic acid C5H9NO4 C00025 5.09 Leucine C6H13NO2 C00123 2.35 Malate C4H6O5 C00149 11.5 nicotinamide adenine dinucleotide C21H28N7O14P2 C00003 9.86 Sedoheptulose 7 phosphate C7H15O10P C05382 7.99 Succinic acid C4H6O4 C00042 11.3 Sucrose C12H22O11 C00089 1.57 Tyrosine C9H11NO3 C00082 3.3 UDP-Glucose C15H24N2O17P2 C00029 11.46 Uridine monophosphate C9H13N2O9P C00105 10.3 METABOLITES_END #END