#METABOLOMICS WORKBENCH kenanazam_20160912_151130_mwtab.txt DATATRACK_ID:737 STUDY_ID:ST000469 ANALYSIS_ID:AN000733 PROJECT_ID:PR000360 VERSION 1 CREATED_ON September 12, 2016, 3:23 pm #PROJECT PR:PROJECT_TITLE Enterococcus faecalis nucleotie profiles PR:PROJECT_SUMMARY Determination of metabolite profiles of E. faecalis strains OG1RF (WT) and PR:PROJECT_SUMMARY rel/relQ (ppGpp0) PR:INSTITUTE Univeristy of Florida PR:DEPARTMENT Oral Biology PR:LABORATORY Oral Biology Lemos-Abranches PR:LAST_NAME Lemos PR:FIRST_NAME Jose PR:ADDRESS 1395 Center Drive, PO Box 100424 Gainesville, FL 32610 PR:EMAIL jlemos@dental.ufl.edu PR:PHONE 352-273-8843 PR:FUNDING_SOURCE UF start-up funds #STUDY ST:STUDY_TITLE Enterococcus faecalis nucleotide profiles - pilot ST:STUDY_TYPE Comparison between wild-type and mutant strain, as well as providing varying ST:STUDY_TYPE cell count input for WT ST:STUDY_SUMMARY E. faecalis OG1RF and rel/relQ will be grown in FMC-AUG to OD600 = 0.25. 3E10 ST:STUDY_SUMMARY cells will be harvested by fast filtration, scraped off of membrane into ST:STUDY_SUMMARY ice-cold PBS, and pelleted by centrifugation. This process will be repeated with ST:STUDY_SUMMARY OG1RF only to harvest 3E9 and 3E8 cells. A second OG1RF sample of 3E10 cells ST:STUDY_SUMMARY will be scraped into formic acid; only acid-soluble extract willl be sent for ST:STUDY_SUMMARY this sample. ST:INSTITUTE Univeristy of Florida ST:DEPARTMENT SECIM ST:LABORATORY Lemos-Abranches ST:LAST_NAME Kajfasz ST:FIRST_NAME Jessica ST:ADDRESS 1395 Center Drive, PO Box 100424 Gainesville, FL 32610 ST:EMAIL jkajfasz@dental.ufl.edu ST:PHONE 352-273-6675 ST:NUM_GROUPS 5 ST:TOTAL_SUBJECTS 5 #SUBJECT SU:SUBJECT_TYPE Bacterial cells SU:SUBJECT_SPECIES Enterococcus faecalis SU:TAXONOMY_ID 1351 SU:GENOTYPE_STRAIN OG1RF #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS Lemos_001 OG1RF_3E10 Strain:WT | Resuspension:PBS CFU=3.00E+10 SUBJECT_SAMPLE_FACTORS Lemos_002 OG1RF_3E9 Strain:WT | Resuspension:PBS CFU=3.00E+09 SUBJECT_SAMPLE_FACTORS Lemos_003 OG1RF_3E8 Strain:WT | Resuspension:PBS CFU=3.00E+08 SUBJECT_SAMPLE_FACTORS Lemos_004 rel/relQ_3E10 Strain:(p)ppGpp0 | Resuspension:PBS CFU=3.00E+10 SUBJECT_SAMPLE_FACTORS Lemos_005 OG1RF_3E10_acid Strain:WT | Resuspension:1M Formic Acid CFU=3.00E+10 #COLLECTION CO:COLLECTION_SUMMARY 3E9, 3E10 - upon reaching OD600 = 0.25, 100mL (OG1RF) or 425mL (rel/relQ) will CO:COLLECTION_SUMMARY be harvested by filtration onto 0.45uM nitrocellulose membrane. The cells will CO:COLLECTION_SUMMARY be scraped off of the membrane into ice-cold PBS or formic acid (~1 mL), CO:COLLECTION_SUMMARY centrifuged for 45 seconds at 12K RPM, liquid aspirated and stored at -80C until CO:COLLECTION_SUMMARY sending for analysis.|3E8 - (OG1RF only). Upon reaching OD600 = 0.25, 1mL CO:COLLECTION_SUMMARY culture will be harvested by 45 seconds centrifugation at 12K RPM, liquid CO:COLLECTION_SUMMARY decanted, and stored at -80C until sending for analysis. CO:SAMPLE_TYPE Bacterial cells #TREATMENT TR:TREATMENT_SUMMARY This pilot study involves no treatment - cells will be grown in chemically TR:TREATMENT_SUMMARY defined medium to an OD600 of 0.25 and harvested for analysis #SAMPLEPREP SP:SAMPLEPREP_SUMMARY NAD|NADH SP:SAMPLEPREP_PROTOCOL_FILENAME SB_NAD-ATP_Assay_Agilent.pdf;SB_NADH_Assay_Agilent.pdf #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY NADH CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 Infinity CH:COLUMN_NAME Waters Acquity HSS T3 (100 x 2.1mm, 1.8um) CH:METHODS_FILENAME SB_NADH_Assay_Agilent.pdf #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6490 QQQ MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS NADH #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS nmol/mg protein MS_METABOLITE_DATA_START Samples OG1RF_3E10 OG1RF_3E9 OG1RF_3E8 rel/relQ_3E10 OG1RF_3E10_acid Factors Strain:WT | Resuspension:PBS Strain:WT | Resuspension:PBS Strain:WT | Resuspension:PBS Strain:(p)ppGpp0 | Resuspension:PBS Strain:WT | Resuspension:1M Formic Acid AMP 0.845457492 0.255958742 0.377771343 ADP 2.377354704 2.260149639 5.589016127 1.694243851 3.516579009 ATP 4.654067744 5.208946868 6.989888429 1.025161287 GDP 0.760383757 0.739643642 0.419042181 0.562394336 GTP 1.286746555 1.413418092 2.632834242 0.218756027 CDP 0.382224859 0.31849744 0.411867 0.655777432 CTP 0.988035124 1.219911451 1.078961586 2.477695127 0.323393839 UDP 0.87831059 0.779262793 1.349374202 0.590703813 1.10056381 UTP 3.223402275 3.796559869 3.217282727 4.902149993 0.763308277 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name retention index quantified m/z PubChem ID KEGG ID AMP 1.51 348.1 6083 ADP 1.51 428 6022 ATP 1.5 508.1 5957 GDP 2.71 444.1 8977 GTP 2.78 524 6830 CDP 1.65 404.1 6132 CTP 1.67 484 6176 UDP 1.72 405 6031 UTP 1.79 485 6133 METABOLITES_END #END