{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST000399","ANALYSIS_ID":"AN000637","VERSION":"1","CREATED_ON":"May 17, 2016, 2:24 pm"},

"PROJECT":{"PROJECT_TITLE":"E.coli effects on growth and substrate uptake of green algae","PROJECT_SUMMARY":"The purpose of this project was to quantify the exchange of thiamine between bacteria and algae. We previously observed that the model bacteria, Escherichia coli, enhanced the growth and substrate uptake of the green algae, Auxenochlorella protothecoides. We hypothesized that this growth enhancement was due to the secretion of thiamine derivatives or degradation products by E. coli followed by uptake of these compounds by A. protothecoides. Targeted and untargeted LCMS revealed the presence of thiamine dervatives in E. coli cell extracts. These LCMS methods were also used to quantify thiamine derivatives and two degradation products, HMP and THZ, present in E. coli medium after cell removal. The LCMS results along with culture studies were employed to show that thiamine derivatives and degradation products were the primary mechanism of symbiosis between E. coli and A. protothecoides.","INSTITUTE":"University of California, Davis","DEPARTMENT":"Genome and Biomedical Sciences Facility","LABORATORY":"WCMC Metabolomics Core","LAST_NAME":"Fiehn","FIRST_NAME":"Oliver","ADDRESS":"1315 Genome and Biomedical Sciences Facility, 451 Health Sciences Drive, Davis, CA 95616","EMAIL":"ofiehn@ucdavis.edu","PHONE":"(530) 754-8258","FUNDING_SOURCE":"NIH U24DK097154"},

"STUDY":{"STUDY_TITLE":"E.coli effects on growth and substrate uptake of green algae (part I - HILIC)","STUDY_SUMMARY":"The purpose of this project was to quantify the exchange of thiamine between bacteria and algae. We previously observed that the model bacteria, Escherichia coli, enhanced the growth and substrate uptake of the green algae, Auxenochlorella protothecoides. We hypothesized that this growth enhancement was due to the secretion of thiamine derivatives or degradation products by E. coli followed by uptake of these compounds by A. protothecoides. Targeted and untargeted LCMS revealed the presence of thiamine dervatives in E. coli cell extracts. These LCMS methods were also used to quantify thiamine derivatives and two degradation products, HMP and THZ, present in E. coli medium after cell removal. The LCMS results along with culture studies were employed to show that thiamine derivatives and degradation products were the primary mechanism of symbiosis between E. coli and A. protothecoides.","INSTITUTE":"University of California, Davis","DEPARTMENT":"Genome and Biomedical Sciences Facility","LABORATORY":"WCMC Metabolomics Core","LAST_NAME":"Fiehn","FIRST_NAME":"Oliver","ADDRESS":"1315 Genome and Biomedical Sciences Facility, 451 Health Sciences Drive, Davis, CA 95616","EMAIL":"ofiehn@ucdavis.edu","PHONE":"(530) 754-8258"},

"SUBJECT":{"SUBJECT_TYPE":"Cells","SUBJECT_SPECIES":"Escherichia coli","TAXONOMY_ID":"562"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"BH28",
"Sample ID":"BH28_HILIC.d",
"Factors":{"Type of media samples":"Base Degraded Thiamine"}
},
{
"Subject ID":"BH29",
"Sample ID":"BH29_HILIC.d",
"Factors":{"Type of media samples":"Fractionated using RP column"}
},
{
"Subject ID":"BH30",
"Sample ID":"BH30_HILIC.d",
"Factors":{"Type of media samples":"Fractionated using RP column"}
},
{
"Subject ID":"BH31",
"Sample ID":"BH31_HILIC.d",
"Factors":{"Type of media samples":"Fractionated using RP column"}
},
{
"Subject ID":"BH32",
"Sample ID":"BH32_HILIC.d",
"Factors":{"Type of media samples":"Fractionated using RP column"}
},
{
"Subject ID":"BH33",
"Sample ID":"BH33_HILIC.d",
"Factors":{"Type of media samples":"Fractionated using RP column"}
},
{
"Subject ID":"BH34",
"Sample ID":"BH34_HILIC.d",
"Factors":{"Type of media samples":"Fractionated using RP column"}
},
{
"Subject ID":"BH35",
"Sample ID":"BH35_HILIC.d",
"Factors":{"Type of media samples":"Methanol Extracted"}
},
{
"Subject ID":"BH36",
"Sample ID":"BH36_HILIC.d",
"Factors":{"Type of media samples":"Methanol Extracted"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Cultures were harvested after 5 days of growth, washed with dH2O, and freeze dried 5 ml of trichloroacetic acid was added to entire freeze dried cell pellet in 15 ml tube, vortexed, and incubated on shaker at 150 rpm for 3 hours TCA was extracted twice w/ 5ml diethyl ether, then sample was washed with 5 ml chloroform, and aqueous phase was recovered Freeze-dried aqueous phase, resuspended in 1 ml MeOH and incubated on shaker for 1 hour at 150 rpm, centrifuged Supernatant was trasnferred to 2 ml tube, sample washed with additional 0.5 ml MeOH and added to 2 ml tube Freeze dried. Note that a method blank and 2 recovery standards containing thiamine were included in this processing","COLLECTION_PROTOCOL_FILENAME":"Thiamine_BH_IG032715QTrap.xlsx","SAMPLE_TYPE":"Cell"},

"TREATMENT":{"TREATMENT_SUMMARY":"BH28 was base degraded thiamine. It did not come from an organism. It was suspended thiamine in strong base for an extended period of time. This was done to see what types of degraded products would form. BH29-34 were E. coli medium samples that were fractionated using a reverse phase column. BH35-36 were media samples after E. coli growth where methanol was used for extraction."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"1. Add 0.5mL of extraction solvent to tube, gently pipet to remove all cells, transfer cells to 2mL eppendorf tube. Repeat for a total of 1mL extraction solvent + cells in 2mL eppendorf tube. 2. Add 2 small stainless steel grinding beads to eppendorf tube 3. Use the GenoGrinder to grind for 3 minutes at 1,250 rpm. 4. Centrifuge at 14,000xg for 5 minutes. 5. Transfer supernatant to a fresh 2mL eppendorf tube. 6. Add 1mL of extraction solvent to tube containing cell pellet + beads, and repeat steps 3 and 4. 7. Collect supernatant, and combine with supernatant collected in step 5. Total volume of extracted sample will be approximately 2mL. 8. Dry down 50uL of extracted sample in 1.5mL eppendorf tube for GC-TOF analysis. 9. Store backups in -20 or -80C.","SAMPLEPREP_PROTOCOL_FILENAME":"SOP_Extraction_of_Yeast_Cells.pdf"},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"HILIC","INSTRUMENT_NAME":"Agilent 6550","COLUMN_NAME":"Waters Acquity BEH Amide (150 x 2.1mm, 1.7um) Waters Acquity","FLOW_GRADIENT":"100% B to 30% B","FLOW_RATE":"0.4 mL/min","COLUMN_TEMPERATURE":"45 C","METHODS_FILENAME":"Data_Dictionary_Fiehn_laboratory_HILIC_QTOF_MS.pdf","SOLVENT_A":"Ultrapure Ultrapure water with 10 mM ammonium formiate + 0.125% formic acid, pH 3","SOLVENT_B":"95:5 v/v acetonitrile:ultrapure water w/ 10 mM ammonium formiate + 0.125% formic acid, pH 3","COLUMN_PRESSURE":"200-700 bar","INJECTION_TEMPERATURE":"4 C","INTERNAL_STANDARD":"See data dictionary","RETENTION_TIME":"See data dictionary","SAMPLE_INJECTION":"3uL","ANALYTICAL_TIME":"14 min","CAPILLARY_VOLTAGE":"4500 V","TIME_PROGRAM":"16.75 min","WEAK_WASH_SOLVENT_NAME":"1:1 ACN:H2O","STRONG_WASH_SOLVENT_NAME":"1:1 ACN:H2O","TARGET_SAMPLE_TEMPERATURE":"Autosampler temp 4 C","RANDOMIZATION_ORDER":"Excel generated"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS","LABORATORY_NAME":"WCMC Metabolomics Core","SOFTWARE_VERSION":"ChromaTOF","DATA_FORMAT":".d"},

"MS":{"MS_COMMENTS":"-","INSTRUMENT_NAME":"Agilent 6550 QTOF","INSTRUMENT_TYPE":"QTOF","MS_TYPE":"ESI","ION_MODE":"NEGATIVE","CAPILLARY_VOLTAGE":"3500 V","COLLISION_GAS":"Nitrogen","DRY_GAS_FLOW":"13 L/min","DRY_GAS_TEMP":"200 C","FRAGMENT_VOLTAGE":"175 V","FRAGMENTATION_METHOD":"Auto MS/MS","ION_SOURCE_TEMPERATURE":"325 C","ION_SPRAY_VOLTAGE":"1000 V","IONIZATION":"Neg","PRECURSOR_TYPE":"Intact Molecule","REAGENT_GAS":"Nitrogen","SOURCE_TEMPERATURE":"325 C","DATAFORMAT":".d","DESOLVATION_GAS_FLOW":"11 L/min","DESOLVATION_TEMPERATURE":"350 C","NEBULIZER":"35 psig","OCTPOLE_VOLTAGE":"750 V","RESOLUTION_SETTING":"extended dynamic range","SCAN_RANGE_MOVERZ":"60-1700 Da","SCANNING_CYCLE":"2 Hz","SCANNING_RANGE":"60-1700 Da","SKIMMER_VOLTAGE":"1850 V"},

"MS_METABOLITE_DATA":{
"Units":"counts",

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