#METABOLOMICS WORKBENCH ankita_pal_20220202_062900_mwtab.txt DATATRACK_ID:3059 STUDY_ID:ST002076 ANALYSIS_ID:AN003384 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 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 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 alpha-Ketoglutarate 2.431658199 1.789205951 2.116091928 2.066433109 2.065677459 2.033558554 0.645455877 0.670380777 0.810876123 0.688494656 0.718399225 0.741767601 5.451844176 4.349768495 3.118201654 6.99356292 3.339051315 3.815928868 2.353047994 2.131632308 1.53433966 2.415531879 2.798605127 1.300210696 Fructose-6-Phosphate 5.423763876 4.405486791 16.3142903 16.01143151 11.56342273 7.43821452 3.548670156 5.097620367 3.226504662 3.34108056 4.437055681 2.943447491 17.14735864 4.304808135 4.06002902 16.95621135 4.24102909 3.412656486 17.70890523 4.740773527 4.351307618 4.158460711 16.65900464 4.52662528 Ribose-5-Phosphate 0.032151329 0.033917529 0.007234757 0.006565163 0.009298473 0.03248734 0.087262281 0.009336461 0.007318936 0.104591166 0.010930377 0.008211962 0.075553007 0.027247941 0.027289401 0.073286812 0.026333036 0.026723389 0.123031421 0.031658902 0.04333407 0.033049069 0.137606392 0.041960035 Malate 19.90638065 21.43032129 73.1064053 74.62490908 70.69678736 36.06239724 34.81226503 41.13542881 57.25997478 34.01959798 41.74953429 40.80929187 289.5362847 66.20505059 68.69375201 214.772623 60.51750289 49.55848354 316.6736039 94.76669124 55.71567261 102.6653924 214.2011088 71.19731941 Glucose-6-Phosphate 13.55050768 14.04397965 29.99861101 30.35839114 26.21402194 19.88002931 9.172061412 12.15652294 9.093103851 9.541828301 12.59363154 8.871851162 40.83291917 8.295110796 4.526320319 40.04789451 8.478892436 4.506587429 42.14467869 9.771357961 9.080480875 9.694708739 38.52490195 9.159506135 Phosphoenolpyruvate 4.798302669 4.894499749 3.255626549 3.303384229 2.099187447 7.255092022 6.957524323 6.779696933 8.594360489 6.955638168 6.638804464 8.60501488 4.777013053 10.976974 15.47544825 4.910222708 10.69721985 15.21845978 5.482889315 19.41841918 17.77764946 19.00523441 5.299408007 17.68766984 ATP 6.535747233 7.088436002 22.66024116 24.79560275 28.08890445 11.8337033 8.821950864 6.976241976 11.49960381 9.001994269 7.115437722 12.36655837 16.26314977 12.13868745 13.9606097 24.73906658 NA 13.4647948 16.829705 21.39663772 15.40383268 22.77940296 40.45357753 15.13112394 Fumarate 4.787772326 5.053553076 14.04159696 11.87665569 12.89083684 10.79603175 4.990073876 7.45749267 11.12538914 4.605361506 7.441668957 1.564585934 41.65672928 6.509451872 10.97478332 23.76191327 6.145993175 10.12031909 103.9344562 25.55038665 23.80622659 18.18004731 8.142778871 8.266179032 ADP 16.33020003 15.82054574 28.08652004 25.79234697 27.85409634 18.47939606 10.90317923 10.96211187 12.63993921 10.61022077 10.34980479 13.56169469 23.2060676 21.54407296 21.05266699 24.18633955 22.20945593 22.10040325 31.44313876 26.5476842 23.89947915 24.16662705 30.62012497 21.02929955 AMP 21.99569867 21.16336754 19.3854082 19.11392645 21.22883989 21.77028447 12.13399228 10.98541526 13.58359569 12.06911331 10.4213231 14.41029447 21.77355095 48.55258893 42.3862744 24.8010042 47.2199083 43.39773111 27.1860819 34.41300661 43.4843683 33.05995754 30.72670448 38.475703 Glycerate-3-phosphate 0.89684201 0.9045242 0.669196109 0.666518854 0.479202265 1.304808282 0.430995807 0.475604684 0.477289952 0.427815192 0.479060106 0.472759317 1.211825136 0.860321196 0.778685213 1.210638705 0.865132558 0.773263862 1.401317563 1.221568162 1.236765663 1.210419807 1.416808944 1.258792542 Citrate 0.766071741 0.757975285 1.480071035 1.464291765 1.834493128 0.81563741 0.260000778 0.229054813 0.339400878 0.259520854 0.226569304 0.34252183 1.943411865 1.308118093 0.652162399 1.978200368 1.323789535 0.786306135 0.99786812 0.551879205 0.481993416 0.562321795 1.039222309 0.472675714 Fructose-1,6-bisphosphate 0.30287192 0.305494968 1.14130549 1.194830878 0.998739348 0.287761915 0.303983467 0.295826768 0.311870051 0.28489005 0.29394869 0.298914464 1.47209977 0.637781753 0.560905165 1.440010955 0.612959607 0.545452751 1.80988431 0.64503778 0.880307706 0.640489168 1.570475143 0.923816763 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name 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