#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