#METABOLOMICS WORKBENCH ankita_pal_20200707_105152 DATATRACK_ID:2093 STUDY_ID:ST001421 ANALYSIS_ID:AN002377 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 NEGATIVE 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 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 alpha-Ketoglutarate 2.431658199 1.789205951 2.116091928 2.066433109 2.065677459 2.033558554 2.429993156 3.391099069 3.177680424 3.387911086 3.538237628 3.245481926 0.829544739 0.959805731 1.457609705 0.778937342 1.48088701 0.903385226 2.603934021 2.126187199 3.076298958 3.299305605 2.881891128 3.42422741 Fructose-6-Phosphate 5.423763876 4.405486791 16.3142903 16.01143151 11.56342273 7.43821452 4.866289313 9.054552436 8.453459673 7.397773321 6.544550977 7.092597352 9.873654693 11.27054019 6.705771003 6.853134595 6.078578345 5.812600782 15.67634033 15.47928349 5.377538979 7.134389512 5.624949411 7.25057276 Ribose-5-Phosphate 0.032151329 0.033917529 0.007234757 0.006565163 0.009298473 0.03248734 0.111578713 0.067250496 0.115720884 0.022108707 0.111567499 0.020476256 0.105354991 0.48529176 0.01861013 0.019085493 0.019798741 0.019548221 0.238864775 0.547149434 0.010629021 0.052862459 0.012303299 0.054266143 Malate 19.90638065 21.43032129 73.1064053 74.62490908 70.69678736 36.06239724 45.8046737 76.64678524 77.7624672 82.42106298 77.62991073 80.66824177 40.36774333 66.75777945 59.05123077 67.2433264 58.20171711 65.67555016 55.16408269 69.26963683 27.17590753 39.79755306 25.92255528 44.83775376 Glucose-6-Phosphate 13.55050768 14.04397965 29.99861101 30.35839114 26.21402194 19.88002931 17.71823136 46.31491562 15.35884231 17.19343482 15.27561232 16.90252226 34.19543738 35.24641421 17.32736356 18.40872847 17.29459261 18.45831965 37.79266453 54.5335739 7.156886597 16.4017309 7.144200533 17.14129914 Phosphoenolpyruvate 4.798302669 4.894499749 3.255626549 3.303384229 2.099187447 7.255092022 24.08781548 14.97361081 10.86455892 17.50143452 11.06156678 17.07909403 9.062821517 22.77941125 11.38196119 12.99181938 11.18218564 12.97329978 17.57693683 27.8355461 14.29826444 8.328226639 14.39699069 8.464401755 ATP 6.535747233 7.088436002 22.66024116 24.79560275 28.08890445 11.8337033 14.27939868 12.82771414 17.60609598 13.1998636 18.65908439 13.48768573 8.796733738 11.85982115 12.81359408 11.64373418 12.31453677 12.23508837 7.186652858 7.307086425 18.3475663 16.72747755 19.73243754 15.23704587 Fumarate 4.787772326 5.053553076 14.04159696 11.87665569 12.89083684 10.79603175 13.87496623 24.77053752 8.7580148 7.889922881 7.912543072 8.5017445 8.104881975 17.57023001 5.573336451 8.113009705 6.351297168 11.5753983 13.82246815 16.02217763 3.846288236 4.779208372 4.358356439 7.164155964 ADP 16.33020003 15.82054574 28.08652004 25.79234697 27.85409634 18.47939606 11.90526416 17.05328142 45.47420467 22.32398526 25.73457759 23.06437326 14.26267605 16.36783582 22.45570975 18.21832504 22.02157396 19.755701 18.55370501 21.00693638 25.69125161 23.69603139 26.97457273 23.31414869 AMP 21.99569867 21.16336754 19.3854082 19.11392645 21.22883989 21.77028447 6.884771615 10.19877631 30.50924957 54.66057282 28.57443432 52.79313026 14.83394809 9.665784541 31.14205111 28.29451871 29.10457451 28.96422474 20.34606081 19.69665551 40.57162983 53.8916265 40.64513259 43.61091296 Glycerate-3-phosphate 0.89684201 0.9045242 0.669196109 0.666518854 0.479202265 1.304808282 1.32474525 2.039622919 0.790168004 1.08253621 0.802663663 1.067889303 1.658615138 2.040894508 0.976969051 1.027638369 0.986052407 1.02678918 3.270160475 4.16513894 1.018271591 1.394863371 1.014017966 1.357266312 Citrate 0.766071741 0.757975285 1.480071035 1.464291765 1.834493128 0.81563741 0.292628008 0.713626606 0.906245135 0.865353348 0.941852471 0.889714988 0.145356846 0.154645742 0.425899167 0.386459377 0.437474343 0.429439822 0.895758603 0.743040806 1.544427121 1.53681298 1.543176958 1.530377579 Fructose-1,6-bisphosphate 0.30287192 0.305494968 1.14130549 1.194830878 0.998739348 0.287761915 0.100096825 0.16755434 0.483820724 0.877895524 0.483034713 0.829262654 0.421384062 0.200393853 0.848835609 0.726518181 0.828458093 0.727992902 0.295346135 0.289406954 0.630133401 1.344175923 0.619678307 1.344154514 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name quantitated m/z [M-H]- KEGG_id alpha-Ketoglutarate 145.0142 C00026 Ribose-5-Phosphate 229.0118 C00117 Fructose-6-Phosphate 259.0224 C00085 Malate 133.0142 C00149 Glucose-6-Phosphate 259.0224 C00092 Phosphoenolpyruvate 166.975 C00074 Citrate 191.0191 C00158 Fructose-1,6-bisphosphate 338.9887 C00354 Glycerate-3-phosphate 184.9856 C00190 AMP 346.0558 C00020 ADP 426.0221 C00008 ATP 505.9885 C00002 Fumarate 115.0036 C00122 METABOLITES_END #END