#METABOLOMICS WORKBENCH ankita_pal_20200707_105152 DATATRACK_ID:2093 STUDY_ID:ST001421 ANALYSIS_ID:AN002376 PROJECT_ID:PR000975 VERSION 1 CREATED_ON July 10, 2020, 1:50 pm #PROJECT PR:PROJECT_TITLE Metabolomic analysis of Escherichia coli K-12 MG1655 transcriptional regulator PR:PROJECT_TITLE mutants under anaerobic conditions PR:PROJECT_TYPE Metabolomics PR:PROJECT_SUMMARY Metabolomic analysis of Wildtype, fnr, arcA and ihf mutants in glucose minimal PR:PROJECT_SUMMARY media under anaerobic fermentation conditions during its exponential phase of PR:PROJECT_SUMMARY growth. Three biological and two technical replicates samples (n=6) were PR:PROJECT_SUMMARY harvested for each of the strains while growing in a bioreactor anaerobically at PR:PROJECT_SUMMARY 37 degrees Celsius and 150 rpm. This study aims to characterize and compare the PR:PROJECT_SUMMARY metabolic profiles of all these strains. PR:INSTITUTE IIT Bombay PR:DEPARTMENT Department of Chemical Engineering PR:LABORATORY Systems Biology and Metabolic Engineering Laboratory PR:LAST_NAME Pal PR:FIRST_NAME Ankita PR:ADDRESS Department of Chemical Engineering, IIT Bombay, Mumbai, Maharashtra, 400076, PR:ADDRESS India PR:EMAIL pal.ankita90@gmail.com PR:PHONE 08879536816 #STUDY ST:STUDY_TITLE Metabolomic study of Escherichia coli K-12 MG1655 WT and its transcriptional ST:STUDY_TITLE regulator mutants under anaerobic fermentation conditions ST:STUDY_TYPE Metabolomics ST:STUDY_SUMMARY Metabolomic analysis of Wildtype, fnr, arcA and ihf mutants in glucose minimal ST:STUDY_SUMMARY media under anaerobic fermentation conditions during its exponential phase of ST:STUDY_SUMMARY growth. Three biological and two technical replicate samples (n=6) were ST:STUDY_SUMMARY harvested for each of the strains while growing in a bioreactor anaerobically at ST:STUDY_SUMMARY 37 degrees Celsius and 150 rpm. This study aims to characterize and compare the ST:STUDY_SUMMARY metabolic profiles of all these strains. ST:INSTITUTE IIT Bombay ST:DEPARTMENT Department of Chemical Engineering ST:LABORATORY Systems Biology and Metabolic Engineering Laboratory (SBMEL) ST:LAST_NAME Pal ST:FIRST_NAME Ankita ST:ADDRESS Department of Chemical Engineering, IIT Bombay, Mumbai, Maharashtra, 400076, ST:ADDRESS India ST:EMAIL pal.ankita90@gmail.com ST:PHONE 08879536816 #SUBJECT SU:SUBJECT_TYPE Bacteria SU:SUBJECT_SPECIES Escherichia coli SU:TAXONOMY_ID 562 SU:GENOTYPE_STRAIN K-12 MG1655 #FACTORS #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS Wild-type_1 WT_1 Genotype:Wildtype RAW_FILE_NAME=WT_positive_1.RAW;WT_negative_1.RAW SUBJECT_SAMPLE_FACTORS Wild-type_2 WT_2 Genotype:Wildtype RAW_FILE_NAME=WT_positive_2.RAW;WT_negative_2.RAW SUBJECT_SAMPLE_FACTORS Wild-type_3 WT_3 Genotype:Wildtype RAW_FILE_NAME=WT_positive_3.RAW;WT_negative_3.RAW SUBJECT_SAMPLE_FACTORS Wild-type_4 WT_4 Genotype:Wildtype RAW_FILE_NAME=WT_positive_4.RAW;WT_negative_4.RAW SUBJECT_SAMPLE_FACTORS Wild-type_5 WT_5 Genotype:Wildtype RAW_FILE_NAME=WT_positive_5.RAW;WT_negative_5.RAW SUBJECT_SAMPLE_FACTORS Wild-type_6 WT_6 Genotype:Wildtype RAW_FILE_NAME=WT_positive_6.RAW;WT_negative_6.RAW SUBJECT_SAMPLE_FACTORS FNR-knockout_1 FNR_1 Genotype:Mutant RAW_FILE_NAME=FNR_positive_1.RAW;FNR_negative_1.RAW SUBJECT_SAMPLE_FACTORS FNR-knockout_2 FNR_2 Genotype:Mutant RAW_FILE_NAME=FNR_positive_2.RAW;FNR_negative_2.RAW SUBJECT_SAMPLE_FACTORS FNR-knockout_3 FNR_3 Genotype:Mutant RAW_FILE_NAME=FNR_positive_3.RAW;FNR_negative_3.RAW SUBJECT_SAMPLE_FACTORS FNR-knockout_4 FNR_4 Genotype:Mutant RAW_FILE_NAME=FNR_positive_4.RAW;FNR_negative_4.RAW SUBJECT_SAMPLE_FACTORS FNR-knockout_5 FNR_5 Genotype:Mutant RAW_FILE_NAME=FNR_positive_5.RAW;FNR_negative_5.RAW SUBJECT_SAMPLE_FACTORS FNR-knockout_6 FNR_6 Genotype:Mutant RAW_FILE_NAME=FNR_positive_6.RAW;FNR_negative_6.RAW SUBJECT_SAMPLE_FACTORS ArcA-knockout_1 ArcA_1 Genotype:Mutant RAW_FILE_NAME=ArcA_positive_1.RAW;ArcA_negative_1.RAW SUBJECT_SAMPLE_FACTORS ArcA-knockout_2 ArcA_2 Genotype:Mutant RAW_FILE_NAME=ArcA_positive_2.RAW;ArcA_negative_2.RAW SUBJECT_SAMPLE_FACTORS ArcA-knockout_3 ArcA_3 Genotype:Mutant RAW_FILE_NAME=ArcA_positive_3.RAW;ArcA_negative_3.RAW SUBJECT_SAMPLE_FACTORS ArcA-knockout_4 ArcA_4 Genotype:Mutant RAW_FILE_NAME=ArcA_positive_4.RAW;ArcA_negative_4.RAW SUBJECT_SAMPLE_FACTORS ArcA-knockout_5 ArcA_5 Genotype:Mutant RAW_FILE_NAME=ArcA_positive_5.RAW;ArcA_negative_5.RAW SUBJECT_SAMPLE_FACTORS ArcA-knockout_6 ArcA_6 Genotype:Mutant RAW_FILE_NAME=ArcA_positive_6.RAW;ArcA_negative_6.RAW SUBJECT_SAMPLE_FACTORS IHF-knockout_1 IHF_1 Genotype:Mutant RAW_FILE_NAME=IHF_positive_1.RAW;IHF_negative_1.RAW SUBJECT_SAMPLE_FACTORS IHF-knockout_2 IHF_2 Genotype:Mutant RAW_FILE_NAME=IHF_positive_2.RAW;IHF_negative_2.RAW SUBJECT_SAMPLE_FACTORS IHF-knockout_3 IHF_3 Genotype:Mutant RAW_FILE_NAME=IHF_positive_3.RAW;IHF_negative_3.RAW SUBJECT_SAMPLE_FACTORS IHF-knockout_4 IHF_4 Genotype:Mutant RAW_FILE_NAME=IHF_positive_4.RAW;IHF_negative_4.RAW SUBJECT_SAMPLE_FACTORS IHF-knockout_5 IHF_5 Genotype:Mutant RAW_FILE_NAME=IHF_positive_5.RAW;IHF_negative_5.RAW SUBJECT_SAMPLE_FACTORS IHF-knockout_6 IHF_6 Genotype:Mutant RAW_FILE_NAME=IHF_positive_6.RAW;IHF_negative_6.RAW #COLLECTION CO:COLLECTION_SUMMARY The WT, fnr, arcA and ihf mutants were grown in a bioreactor in glucose minimal CO:COLLECTION_SUMMARY media under anaerobic fermentation conditions at 37 degrees Celsius and 150 rpm. CO:COLLECTION_SUMMARY The samples were harvested during its exponential phase of growth for three CO:COLLECTION_SUMMARY biological and two technical replicates (n=6). The metabolites were extracted CO:COLLECTION_SUMMARY using the Methanol-Chloroform-Water method. Detailed sample collection protocol CO:COLLECTION_SUMMARY is described in “Extraction_protocol_msi.pdf”. CO:COLLECTION_PROTOCOL_FILENAME Extraction_protocol_msi.pdf CO:SAMPLE_TYPE Bacterial cells CO:COLLECTION_METHOD Methanol-Chloroform-Water method CO:COLLECTION_LOCATION IIT Bombay, Powai, Mumbai- 400076, Mumbai, Maharashtra CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY The WT, fnr, arcA and ihf mutants were grown in a bioreactor in glucose minimal TR:TREATMENT_SUMMARY media under anaerobic fermentation conditions at 37 degrees Celsius and 150 rpm. TR:TREATMENT_SUMMARY The samples were harvested during its exponential phase of growth for three TR:TREATMENT_SUMMARY biological and two technical replicates (n=6). The metabolites were extracted TR:TREATMENT_SUMMARY using the Methanol-Chloroform-Water method. TR:CELL_GROWTH_CONTAINER Bioreactor TR:CELL_MEDIA M9 + Glucose TR:CELL_ENVIR_COND 37 degrees Celsius, 150 rpm. anaerobic fermentation #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Samples were extracted from the Wildtype, fnr, arcA and ihf mutants grown in a SP:SAMPLEPREP_SUMMARY bioreactor anaerobically in glucose minimal media at 37 degrees Celsius and 150 SP:SAMPLEPREP_SUMMARY rpm. The metabolites were extracted using the Methanol-Chloroform-Water method. SP:SAMPLEPREP_SUMMARY The samples were spiked with an equal volume of 13C-labelled internal standard SP:SAMPLEPREP_SUMMARY taken from the same batch at an earlier stage to prevent to account for losses SP:SAMPLEPREP_SUMMARY due to metabolite degradation and to enable robust quantification circumventing SP:SAMPLEPREP_SUMMARY the ion-suppression effects. SP:SAMPLEPREP_PROTOCOL_FILENAME MS-method_msi.pdf #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY The detailed chromatography and MS protocol has been described in CH:CHROMATOGRAPHY_SUMMARY "MS-method_msi.pdf" file. CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 CH:COLUMN_NAME SeQuant ZIC- pHILIC (150 x 2.1mm, 5um) CH:METHODS_FILENAME MS-method_msi.pdf #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Plus Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Data acquisition done using parent ion MS intensities. Peak height used for MS:MS_COMMENTS processing. Xcalibur 4.3.73.11 used for data analysis and peak integration. MS:MS_COMMENTS Missing value imputation was done using SVD impute function in MetaboAnalyst. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS umol/gDCW MS_METABOLITE_DATA_START Samples WT_1 WT_2 WT_3 WT_4 WT_5 WT_6 FNR_1 FNR_2 FNR_3 FNR_4 FNR_5 FNR_6 ArcA_1 ArcA_2 ArcA_3 ArcA_4 ArcA_5 ArcA_6 IHF_1 IHF_2 IHF_3 IHF_4 IHF_5 IHF_6 Factors Genotype:Wildtype Genotype:Wildtype Genotype:Wildtype Genotype:Wildtype Genotype:Wildtype Genotype:Wildtype Genotype:Mutant Genotype:Mutant Genotype:Mutant Genotype:Mutant Genotype:Mutant Genotype:Mutant Genotype:Mutant Genotype:Mutant Genotype:Mutant Genotype:Mutant Genotype:Mutant Genotype:Mutant Genotype:Mutant Genotype:Mutant Genotype:Mutant Genotype:Mutant Genotype:Mutant Genotype:Mutant Leucine 4.339392777 4.071342108 4.596626868 4.595376637 5.52 5.18 6.400957329 10.46297463 8.317017177 6.815287827 8.507162434 6.62992152 3.498277807 5.15511304 6.236686214 3.470223217 6.011367319 3.353149384 10.78442774 9.899176408 8.171941777 7.636763439 8.061992735 7.310259808 Phenylalanine 0.663109999 0.656541175 0.631053559 0.631705113 0.73 0.97 0.960255482 1.832949007 0.470839972 0.482867824 0.455445593 0.446287823 0.919374719 1.078995121 0.350754917 0.28562148 0.337612049 0.269234839 1.278624674 1.651430028 0.392353579 0.416453329 0.378744908 0.405724361 Methionine 1.71898291 1.653582903 1.962846801 2.00167123 1.93 2.15 2.284351434 3.177159331 2.243830731 2.075451872 2.241108933 2.050252508 2.245156179 3.435884253 2.92447345 2.247891346 2.689877845 2.210463807 3.714671528 4.467733801 2.848516825 2.608039959 2.819914201 2.546690231 Tryptophan 0.133562342 0.135524122 0.13148099 0.130893034 0.13 0.2 0.155114099 0.163983263 0.134010529 0.133309635 0.130628621 0.120272303 0.149292103 0.160557927 0.071185928 0.082106401 0.066810792 0.077600612 0.335915592 0.329427835 0.151157441 0.137912775 0.140411937 0.127793229 Proline 0.596864506 0.584915861 1.104842009 1.0868342 1.27 0.88 2.197420349 2.758159229 1.129651175 0.931316217 1.035418475 0.90061166 1.317195749 2.200637938 1.537510458 0.732046502 1.502324713 0.71257337 2.632512824 2.575394694 2.155921224 1.764593228 2.086671525 1.73153624 Valine 5.386763361 3.10520573 5.572423982 5.628870515 8.29 4.63 7.59487846 11.24208446 17.04611781 11.00740305 16.65561849 10.72153007 3.300576121 5.776501569 9.57817371 4.482262381 12.54724314 4.224681834 8.082713656 7.263760513 9.240514947 8.029229587 15.17189956 7.670512912 Tyrosine 1.18408796 1.198604713 1.846440277 1.95226411 2.85 1.6 1.26395932 3.809440995 2.884604697 2.587744294 2.809217948 2.668907596 1.151714143 1.629195913 1.637825939 1.130908024 1.563744573 1.146346172 1.986286571 2.375440091 1.661753456 1.871655559 1.503531392 1.739794669 Alanine 23.53978108 23.7608482 39.23017769 38.38480395 44.64 33.01 43.7836075 71.02241188 43.90196622 32.13580673 47.8771668 34.36020668 26.34104268 59.8208647 32.36534191 18.00789491 34.09081958 18.71218693 65.5102225 64.05751276 34.81014983 32.95545951 34.92097713 33.12270166 Threonine 2.163773106 2.197844239 2.695540594 2.679859374 3.31 2.69 4.011864681 4.129708141 7.236579635 6.299573039 6.805043381 5.956167302 2.126623086 3.233085166 4.331037788 2.624980014 4.148659664 2.52664784 6.47694215 7.198727943 7.912591471 7.36602894 7.646783705 6.839066035 Histidine 0.6430585 0.642164429 0.976755049 1.091947178 1.244768712 0.887764829 1.000622276 1.198029266 1.400824615 1.086448589 1.155682003 1.06236661 0.763137851 0.829976551 0.684961912 0.720970329 0.827522861 0.701874868 0.959891528 1.194379873 0.874196811 1.029647466 0.877563873 0.998635402 Glutamine 0.463601549 0.436838633 1.580567461 1.56681558 2.83 0.68 2.756510157 4.814927348 3.418029218 1.197681719 3.615157366 1.178887816 2.642274499 3.664556734 8.833052682 1.609133424 8.569836586 1.466870664 1.073436053 0.816358106 2.734031653 3.082490841 2.585753256 2.752000695 Glycine 8.471052718 8.242268544 8.885631666 9.200894163 9.36 11.86 12.92605469 15.63830643 13.362533 11.73774094 15.95005763 13.89543723 12.47293033 17.24236313 9.048569862 8.856935801 9.814400778 9.168229217 17.77297138 21.02193491 12.35568658 11.55306674 11.13033875 10.56060579 Serine 3.931485656 3.983659613 5.702612629 6.113026786 6.52247969 5.951601445 3.632689301 4.675677081 7.449852881 6.164549639 6.751670376 5.385807885 7.032423266 7.226453385 5.078324867 8.189607009 4.832307204 7.569581312 7.152756093 12.3768043 6.24891575 7.108783413 5.720280041 6.312525658 Asparagine 0.080912663 0.08028805 1.084927517 0.872941862 1.272963641 0.076990564 0.935287859 3.004985272 2.59289939 1.034796462 2.910862817 1.097177447 0.148699026 0.174963177 0.546885805 0.668532603 0.737380438 0.673863079 0.144549282 0.22329632 1.213741775 0.992764859 1.298463567 0.910067671 Glutamate 35.38984382 35.4371323 47.65926338 47.16255016 56.90088362 42.83083614 107.0202249 248.8671366 132.9918981 91.86258608 137.5770703 96.89761181 89.25278514 136.845988 170.8949691 79.38838194 176.5879063 77.79404072 151.2445023 115.9140497 139.1275467 119.761219 133.7223942 115.2004514 Aspartate 1.668348702 1.773337413 1.830172242 1.544621655 2.142711546 3.311893117 4.918719898 12.01578421 5.51112183 3.432252307 5.407146733 3.502383447 6.326100014 19.40505395 8.706339289 10.65151132 8.749116287 9.511270155 9.803389791 9.152478158 3.715692207 2.728806709 3.307037184 2.502641895 Lysine 15.17728428 15.61512655 15.987123 16.22565965 19.81823988 19.95298073 21.32509351 28.00255741 23.77621282 21.2631694 20.88602526 20.68154423 15.7168039 19.71933087 16.7183398 11.40358907 18.59556765 11.13582354 30.36702811 26.35193005 25.37271205 23.19004996 24.26697858 22.73329222 Arginine 3.201064274 3.190855187 2.570239548 2.567657062 2.856752366 4.017516151 3.150330736 3.520520208 4.653681114 4.162921396 4.850216883 4.522572993 4.787272849 5.412203529 4.841560739 3.33436043 4.735368234 3.380225993 5.650059245 4.801431394 5.216369748 5.060006482 5.309176544 5.091251242 NAD 14.50534375 14.33103254 14.97360937 14.64225697 15.23587052 16.65680255 15.21425326 17.14390443 20.14341955 18.73102413 20.94559365 19.25686933 11.96027854 13.14485333 13.9585175 11.68478087 14.04663848 11.61696005 17.80878973 17.53776596 19.08232902 17.96457025 19.30568068 17.89014351 NADPH 0.523640271 0.492833858 0.625037778 0.651290537 0.797642476 0.739486511 0.777625079 0.850576048 0.754848296 0.67265462 0.707140486 0.608821849 0.696914498 0.676645578 0.450921788 0.496730093 0.600171344 0.484768262 0.735193053 0.717657699 0.665553667 0.673228334 0.63678474 0.617294615 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name quantitated m/z [M+H]+ KEGG_id Leucine 132.1019 C00123 Phenylalanine 166.0862 C00079 Methionine 150.0583 C00073 Tryptophan 205.0971 C00078 Proline 116.0706 C00148 Valine 118.0862 C00183 Tyrosine 182.0811 C00082 Alanine 90.0549 C00041 Threonine 120.0655 C00188 Glutamine 147.0764 C00064 Glycine 76.0393 C00037 Aspartate 134.0447 C00049 Asparagine 133.0607 C00152 Serine 106.0498 C00065 Glutamate 148.0604 C00025 Lysine 147.1128 C00047 Arginine 175.1189 C00062 Histidine 156.0767 C00135 NAD 664.1163 C00003 NADPH 746.0983 C00005 METABOLITES_END #END