#METABOLOMICS WORKBENCH ankita_pal_20220202_062900_mwtab.txt DATATRACK_ID:3059 STUDY_ID:ST002076 ANALYSIS_ID:AN003383 PROJECT_ID:PR001316 VERSION 1 CREATED_ON February 2, 2022, 1:22 pm #PROJECT PR:PROJECT_TITLE Metabolomic analysis of Escherichia coli K-12 MG1655 transcriptional regulator PR:PROJECT_TITLE double mutants under anaerobic conditions PR:PROJECT_TYPE Metabolomics PR:PROJECT_SUMMARY Metabolomic analysis of Wildtype, fnr_arcA (FA), arcA_ihf (AI) and fnr_ihf (FI) PR:PROJECT_SUMMARY mutants in glucose minimal media under anaerobic fermentation conditions during PR:PROJECT_SUMMARY its exponential phase of growth. Three biological and two technical replicates PR:PROJECT_SUMMARY samples (n=6) were harvested for each of the strains while growing in a PR:PROJECT_SUMMARY bioreactor anaerobically at 37 degrees Celsius and 150 rpm. This study aims to PR:PROJECT_SUMMARY characterize and compare the 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 double mutants under anaerobic fermentation conditions ST:STUDY_TYPE Metabolomics ST:STUDY_SUMMARY Metabolomic analysis of Wildtype, fnr_arcA (FA), arcA_ihf (AI) and fnr_ihf (FI) ST:STUDY_SUMMARY mutants in glucose minimal media under anaerobic fermentation conditions during ST:STUDY_SUMMARY its exponential phase of growth. Three biological and two technical replicate ST:STUDY_SUMMARY samples (n=6) were harvested for each of the strains while growing in a ST:STUDY_SUMMARY bioreactor anaerobically at 37 degrees Celsius and 150 rpm. This study aims to ST:STUDY_SUMMARY characterize and compare the 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 | KO Type:Wildtype RAW_FILE_NAME=WT_positive_1.RAW;WT_negative_1.RAW SUBJECT_SAMPLE_FACTORS Wild-type_2 WT_2 Genotype:Wildtype | KO Type:Wildtype RAW_FILE_NAME=WT_positive_2.RAW;WT_negative_2.RAW SUBJECT_SAMPLE_FACTORS Wild-type_3 WT_3 Genotype:Wildtype | KO Type:Wildtype RAW_FILE_NAME=WT_positive_3.RAW;WT_negative_3.RAW SUBJECT_SAMPLE_FACTORS Wild-type_4 WT_4 Genotype:Wildtype | KO Type:Wildtype RAW_FILE_NAME=WT_positive_4.RAW;WT_negative_4.RAW SUBJECT_SAMPLE_FACTORS Wild-type_5 WT_5 Genotype:Wildtype | KO Type:Wildtype RAW_FILE_NAME=WT_positive_5.RAW;WT_negative_5.RAW SUBJECT_SAMPLE_FACTORS Wild-type_6 WT_6 Genotype:Wildtype | KO Type:Wildtype RAW_FILE_NAME=WT_positive_6.RAW;WT_negative_6.RAW SUBJECT_SAMPLE_FACTORS FA-knockout_1 FA_1 Genotype:Mutant | KO Type:FA RAW_FILE_NAME=FA_positive_1.RAW;FA_negative_1.RAW SUBJECT_SAMPLE_FACTORS FA-knockout_2 FA_2 Genotype:Mutant | KO Type:FA RAW_FILE_NAME=FA_positive_2.RAW;FA_negative_2.RAW SUBJECT_SAMPLE_FACTORS FA-knockout_3 FA_3 Genotype:Mutant | KO Type:FA RAW_FILE_NAME=FA_positive_3.RAW;FA_negative_3.RAW SUBJECT_SAMPLE_FACTORS FA-knockout_4 FA_4 Genotype:Mutant | KO Type:FA RAW_FILE_NAME=FA_positive_4.RAW;FA_negative_4.RAW SUBJECT_SAMPLE_FACTORS FA-knockout_5 FA_5 Genotype:Mutant | KO Type:FA RAW_FILE_NAME=FA_positive_5.RAW;FA_negative_5.RAW SUBJECT_SAMPLE_FACTORS FA-knockout_6 FA_6 Genotype:Mutant | KO Type:FA RAW_FILE_NAME=FA_positive_6.RAW;FA_negative_6.RAW SUBJECT_SAMPLE_FACTORS AI-knockout_1 AI_1 Genotype:Mutant | KO Type:AI RAW_FILE_NAME=AI_positive_1.RAW;AI_negative_1.RAW SUBJECT_SAMPLE_FACTORS AI-knockout_2 AI_2 Genotype:Mutant | KO Type:AI RAW_FILE_NAME=AI_positive_2.RAW;AI_negative_2.RAW SUBJECT_SAMPLE_FACTORS AI-knockout_3 AI_3 Genotype:Mutant | KO Type:AI RAW_FILE_NAME=AI_positive_3.RAW;AI_negative_3.RAW SUBJECT_SAMPLE_FACTORS AI-knockout_4 AI_4 Genotype:Mutant | KO Type:AI RAW_FILE_NAME=AI_positive_4.RAW;AI_negative_4.RAW SUBJECT_SAMPLE_FACTORS AI-knockout_5 AI_5 Genotype:Mutant | KO Type:AI RAW_FILE_NAME=AI_positive_5.RAW;AI_negative_5.RAW SUBJECT_SAMPLE_FACTORS AI-knockout_6 AI_6 Genotype:Mutant | KO Type:AI RAW_FILE_NAME=AI_positive_6.RAW;AI_negative_6.RAW SUBJECT_SAMPLE_FACTORS FI-knockout_1 FI_1 Genotype:Mutant | KO Type:FI RAW_FILE_NAME=FI_positive_1.RAW;FI_negative_1.RAW SUBJECT_SAMPLE_FACTORS FI-knockout_2 FI_2 Genotype:Mutant | KO Type:FI RAW_FILE_NAME=FI_positive_2.RAW;FI_negative_2.RAW SUBJECT_SAMPLE_FACTORS FI-knockout_3 FI_3 Genotype:Mutant | KO Type:FI RAW_FILE_NAME=FI_positive_3.RAW;FI_negative_3.RAW SUBJECT_SAMPLE_FACTORS FI-knockout_4 FI_4 Genotype:Mutant | KO Type:FI RAW_FILE_NAME=FI_positive_4.RAW;FI_negative_4.RAW SUBJECT_SAMPLE_FACTORS FI-knockout_5 FI_5 Genotype:Mutant | KO Type:FI RAW_FILE_NAME=FI_positive_5.RAW;FI_negative_5.RAW SUBJECT_SAMPLE_FACTORS FI-knockout_6 FI_6 Genotype:Mutant | KO Type:FI RAW_FILE_NAME=FI_positive_6.RAW;FI_negative_6.RAW #COLLECTION CO:COLLECTION_SUMMARY The WT, FA, AI and FI 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, FA, AI and FI 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, FA, AI and FI 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 Method_msi.pdf #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY The detailed chromatography and MS protocol has been described in CH:CHROMATOGRAPHY_SUMMARY "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 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 and Height ratio/gDCW MS_METABOLITE_DATA_START Samples WT_1 WT_2 WT_3 WT_4 WT_5 WT_6 FA_1 FA_2 FA_3 FA_4 FA_5 FA_6 FI_1 FI_2 FI_3 FI_4 FI_5 FI_6 AI_1 AI_2 AI_3 AI_4 AI_5 AI_6 Factors Genotype:Wildtype | KO Type:Wildtype Genotype:Wildtype | KO Type:Wildtype Genotype:Wildtype | KO Type:Wildtype Genotype:Wildtype | KO Type:Wildtype Genotype:Wildtype | KO Type:Wildtype Genotype:Wildtype | KO Type:Wildtype Genotype:Mutant | KO Type:FA Genotype:Mutant | KO Type:FA Genotype:Mutant | KO Type:FA Genotype:Mutant | KO Type:FA Genotype:Mutant | KO Type:FA Genotype:Mutant | KO Type:FA Genotype:Mutant | KO Type:FI Genotype:Mutant | KO Type:FI Genotype:Mutant | KO Type:FI Genotype:Mutant | KO Type:FI Genotype:Mutant | KO Type:FI Genotype:Mutant | KO Type:FI Genotype:Mutant | KO Type:AI Genotype:Mutant | KO Type:AI Genotype:Mutant | KO Type:AI Genotype:Mutant | KO Type:AI Genotype:Mutant | KO Type:AI Genotype:Mutant | KO Type:AI Leucine 4.339392777 4.071342108 4.596626868 4.595376637 5.52 5.18 1.334135164 1.142967846 1.651358914 1.327865207 1.182408213 1.660768564 13.13477172 9.708554001 7.844353096 13.34449652 9.981734138 8.064252229 8.033331143 6.387733342 4.327148457 6.326741784 7.97885782 4.382174085 Phenylalanine 0.663109999 0.656541175 0.631053559 0.631705113 0.73 0.97 0.249103815 0.211276351 0.304333072 0.24610561 0.375756323 0.313151704 0.956369382 0.413428159 0.450790316 0.943536605 0.415352214 0.463092186 1.039098337 0.460916111 0.32387146 0.456135049 1.06090185 0.327080822 Methionine 1.71898291 1.653582903 1.962846801 2.00167123 1.93 2.15 0.809105347 0.744985835 0.936326646 0.823942542 0.656912025 0.964997275 2.596011115 2.280334375 2.103459035 2.572091412 2.309892442 2.059355598 2.216351743 2.387191458 2.279863547 2.52827834 2.236288269 2.241145615 Tryptophan 0.133562342 0.135524122 0.13148099 0.130893034 0.13 0.2 0.051499636 0.049692294 0.066156015 0.055456579 0.044874762 0.063890949 0.202518088 0.146312025 0.138886819 0.204197166 0.143898154 0.144802108 0.207042205 0.169314626 0.102786818 0.17051645 0.245702228 0.116702147 Proline 0.596864506 0.584915861 1.104842009 1.0868342 1.27 0.88 0.400966594 0.261354198 0.582431191 0.398185198 0.251919237 0.592504798 1.736433064 1.588997552 1.376301542 1.746776693 1.552134071 1.398536279 1.339420586 1.502309697 1.133164846 1.448321534 1.391743901 1.080259513 Valine 5.386763361 3.10520573 5.572423982 5.628870515 8.29 4.63 1.169024584 0.994124749 1.451451436 1.160306247 1.101671581 1.479356255 13.25549206 10.77838939 11.33446195 13.71297705 16.33583116 11.33608115 7.399753537 7.160023033 3.896128804 6.964498972 7.363162506 4.012660142 Tyrosine 1.18408796 1.198604713 1.846440277 1.95226411 2.85 1.6 0.688484691 0.609683335 0.834538669 0.674679286 0.844092749 0.815284925 2.05756395 1.341043262 1.607711998 2.18447674 1.305745163 1.483789946 1.966238912 1.24833081 0.868799448 1.268291745 1.849540016 0.878637368 Alanine 23.53978108 23.7608482 39.23017769 38.38480395 44.64 33.01 11.09705196 14.40442051 10.215565 11.33743009 32.006788 10.27019811 51.35264341 29.33835716 29.15341321 55.25011896 30.60755955 30.27611738 36.96062397 24.01924677 16.48727101 23.03208426 37.45738569 17.32539386 Threonine 2.163773106 2.197844239 2.695540594 2.679859374 3.31 2.69 1.003416315 0.774235989 1.337012406 1.020482931 0.523302155 1.318657568 7.352846629 8.476907083 8.109597865 7.555817018 8.923354349 7.814753122 4.314708604 5.015911955 4.209758247 5.287019882 3.968316975 4.238999661 Histidine 0.6430585 0.642164429 0.976755049 1.091947178 1.244768712 0.887764829 0.480039502 0.445725835 0.604561256 0.472007288 0.424618584 0.624710337 1.27 0.98 1.02 NA NA 0.773012391 1.283088491 1.106460705 0.824720654 1.048230798 1.386763257 0.71822009 Glutamine 0.463601549 0.436838633 1.580567461 1.56681558 2.83 0.68 1.198375254 0.531566011 1.946179442 1.123691571 0.4027878 1.932088688 4.123833534 5.219206702 2.256411571 4.500606506 5.491305112 2.263248441 3.839001618 5.426154434 4.986428622 5.289201944 3.805365794 5.100114458 Glycine 8.471052718 8.242268544 8.885631666 9.200894163 9.36 11.86 5.672243972 6.134059287 5.808904945 5.449977698 6.243765901 5.944720401 12.60362145 10.13031634 9.944715887 11.99349803 11.45830145 10.15562456 9.587720086 9.134640886 7.61795928 9.183586026 9.774123118 8.030632387 Serine 3.931485656 3.983659613 5.702612629 6.113026786 6.52247969 5.951601445 3.771216435 3.300305058 4.556059306 3.633197807 3.281372259 4.235544666 7.034895717 6.07731881 5.275624112 5.995693538 5.996232873 5.112533214 10.39618612 11.45672079 7.236180924 11.87117691 10.55521518 7.467889646 Asparagine 0.080912663 0.08028805 1.084927517 0.872941862 1.272963641 0.076990564 0.596646647 0.519423728 0.702530706 0.600692996 0.365497949 0.583284478 1.051949844 1.413520058 0.759372982 1.187664172 1.42657615 0.917921429 3.032687323 0.945376282 0.696285242 0.963146153 3.026555421 0.668639426 Glutamate 35.38984382 35.4371323 47.65926338 47.16255016 56.90088362 42.83083614 40.7892424 30.10862297 52.11396594 40.40062771 28.39434272 51.1250243 180.2213361 166.8578447 127.6251226 217.0593375 176.4325318 136.9200022 215.4303849 199.3880864 139.048606 190.0676802 221.0248281 134.7082602 Aspartate 1.668348702 1.773337413 1.830172242 1.544621655 2.142711546 3.311893117 2.46995608 1.863674066 3.015723455 2.369954652 1.113196337 2.962233157 2.735995272 4.403985356 2.196360864 2.892779605 4.587577196 2.24477211 4.441363884 11.8722842 10.75836206 11.20449343 4.346266355 10.605516 Lysine 15.17728428 15.61512655 15.987123 16.22565965 19.81823988 19.95298073 7.315249483 5.909528117 9.543245747 7.227878962 5.718945131 9.51607613 27.17537491 22.94924859 21.23205314 24.43770328 21.74872394 20.05510147 23.03475856 21.4520317 17.07263614 21.4625641 23.09324813 16.41024413 Arginine 3.201064274 3.190855187 2.570239548 2.567657062 2.856752366 4.017516151 2.123937379 1.771744333 2.80122412 2.156409808 1.321718957 2.811148258 4.915660066 4.985660664 4.063997742 4.935120258 4.931470521 4.065967409 4.372042981 4.977456362 3.710968633 5.040338104 4.162525434 3.717987974 NAD 14.50534375 14.33103254 14.97360937 14.64225697 15.23587052 16.65680255 7.38447865 6.767811734 9.359049145 7.332635571 6.639439134 9.409666066 18.75345166 17.02690229 17.60288283 19.64725485 17.53289881 18.15669169 16.93406411 17.26679837 15.03060162 17.3199791 17.03085487 15.31096649 NADPH 0.523640271 0.492833858 0.625037778 0.651290537 0.797642476 0.739486511 0.369522508 0.322234406 0.45185508 0.370111796 0.10526293 0.452310503 0.691723737 0.550166881 0.594680257 NA NA NA 0.586918908 0.513756159 0.445295753 0.49843337 0.574081836 0.406857981 GABA 0.040205062 0.0446408 0.045472766 0.030308431 0.030026763 0.036162541 0.091145917 0.08278234 0.120162407 0.091828658 0.092596741 0.11538322 0.784288432 0.514689284 0.482440582 0.806118323 0.508669526 0.45019188 0.374969422 0.206166245 0.143975601 0.205740282 0.380080982 0.149087161 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name 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 GABA 104.0706 C00334 METABOLITES_END #END