#METABOLOMICS WORKBENCH kenanazam_20160912_151130_mwtab.txt DATATRACK_ID:737 STUDY_ID:ST000469 ANALYSIS_ID:AN000732 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 NAD CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 Infinity CH:COLUMN_NAME Thermo Hypercarb (50 x 2.1mm, 3um) CH:METHODS_FILENAME SB_NAD-ATP_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 NAD #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 NAM NMN 0.221951536 0.075655737 0.169181763 0.009625221 NAD 3.232703525 3.990031589 5.121248885 3.529679145 2.037619576 NADP 0.173755747 0.230457596 NADH 1.225075661 0.04577082 0.080460281 1.379111802 0.002979648 NADPH MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name retention index quantified m/z PubChem ID KEGG ID NAM 1.69 123 936 NMN 1.25 335.2 14180 NAD 1.92 664.2 5893 NADP 1.7 744.2 5886 NADH 1.86 666.2 439153 NADPH 0.66 746.2 5884 METABOLITES_END #END