#METABOLOMICS WORKBENCH michaelsa93_20160513_094817_mwtab.txt DATATRACK_ID:629 STUDY_ID:ST000400 ANALYSIS_ID:AN001818 PROJECT_ID:PR000312
VERSION 1
CREATED_ON January 9, 2019, 7:22 pm
#PROJECT
PR:PROJECT_TITLE E.coli effects on growth and substrate uptake of green algae
PR:PROJECT_SUMMARY The purpose of this project was to quantify the exchange of thiamine between
PR:PROJECT_SUMMARY bacteria and algae. We previously observed that the model bacteria, Escherichia
PR:PROJECT_SUMMARY coli, enhanced the growth and substrate uptake of the green algae,
PR:PROJECT_SUMMARY Auxenochlorella protothecoides. We hypothesized that this growth enhancement was
PR:PROJECT_SUMMARY due to the secretion of thiamine derivatives or degradation products by E. coli
PR:PROJECT_SUMMARY followed by uptake of these compounds by A. protothecoides. Targeted and
PR:PROJECT_SUMMARY untargeted LCMS revealed the presence of thiamine dervatives in E. coli cell
PR:PROJECT_SUMMARY extracts. These LCMS methods were also used to quantify thiamine derivatives and
PR:PROJECT_SUMMARY two degradation products, HMP and THZ, present in E. coli medium after cell
PR:PROJECT_SUMMARY removal. The LCMS results along with culture studies were employed to show that
PR:PROJECT_SUMMARY thiamine derivatives and degradation products were the primary mechanism of
PR:PROJECT_SUMMARY symbiosis between E. coli and A. protothecoides.
PR:INSTITUTE University of California, Davis
PR:DEPARTMENT Genome and Biomedical Sciences Facility
PR:LABORATORY WCMC Metabolomics Core
PR:LAST_NAME Fiehn
PR:FIRST_NAME Oliver
PR:ADDRESS Health Sciences Drive, Davis, California, 95616, USA
PR:EMAIL ofiehn@ucdavis.edu
PR:PHONE (530) 754-8258
PR:FUNDING_SOURCE NIH U24DK097154
#STUDY
ST:STUDY_TITLE E.coli effects on growth and substrate uptake of green algae (part II - Reverse
ST:STUDY_TITLE Phase)
ST:STUDY_SUMMARY The purpose of this project was to quantify the exchange of thiamine between
ST:STUDY_SUMMARY bacteria and algae. We previously observed that the model bacteria, Escherichia
ST:STUDY_SUMMARY coli, enhanced the growth and substrate uptake of the green algae,
ST:STUDY_SUMMARY Auxenochlorella protothecoides. We hypothesized that this growth enhancement was
ST:STUDY_SUMMARY due to the secretion of thiamine derivatives or degradation products by E. coli
ST:STUDY_SUMMARY followed by uptake of these compounds by A. protothecoides. Targeted and
ST:STUDY_SUMMARY untargeted LCMS revealed the presence of thiamine dervatives in E. coli cell
ST:STUDY_SUMMARY extracts. These LCMS methods were also used to quantify thiamine derivatives and
ST:STUDY_SUMMARY two degradation products, HMP and THZ, present in E. coli medium after cell
ST:STUDY_SUMMARY removal. The LCMS results along with culture studies were employed to show that
ST:STUDY_SUMMARY thiamine derivatives and degradation products were the primary mechanism of
ST:STUDY_SUMMARY symbiosis between E. coli and A. protothecoides.
ST:INSTITUTE University of California, Davis
ST:DEPARTMENT Genome and Biomedical Sciences Facility
ST:LABORATORY WCMC Metabolomics Core
ST:LAST_NAME Fiehn
ST:FIRST_NAME Oliver
ST:ADDRESS Health Sciences Drive, Davis, California, 95616, USA
ST:EMAIL ofiehn@ucdavis.edu
ST:PHONE (530) 754-8258
#SUBJECT
SU:SUBJECT_TYPE Cells
SU:SUBJECT_SPECIES Escherichia coli
SU:TAXONOMY_ID 562
#SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data
SUBJECT_SAMPLE_FACTORS BH28 BH28_RP.d Type of media samples:Base Degraded Thiamine
SUBJECT_SAMPLE_FACTORS BH29 BH29_RP.d Type of media samples:Fractionated using RP column
SUBJECT_SAMPLE_FACTORS BH30 BH30_RP.d Type of media samples:Fractionated using RP column
SUBJECT_SAMPLE_FACTORS BH31 BH31_RP.d Type of media samples:Fractionated using RP column
SUBJECT_SAMPLE_FACTORS BH32 BH32_RP.d Type of media samples:Fractionated using RP column
SUBJECT_SAMPLE_FACTORS BH33 BH33_RP.d Type of media samples:Fractionated using RP column
SUBJECT_SAMPLE_FACTORS BH34 BH34_RP.d Type of media samples:Fractionated using RP column
SUBJECT_SAMPLE_FACTORS BH35 BH35_RP.d Type of media samples:Methanol Extracted
SUBJECT_SAMPLE_FACTORS BH36 BH36_RP.d Type of media samples:Methanol Extracted
#COLLECTION
CO:COLLECTION_SUMMARY Cultures were harvested after 5 days of growth, washed with dH2O, and freeze
CO:COLLECTION_SUMMARY dried 5 ml of trichloroacetic acid was added to entire freeze dried cell pellet
CO:COLLECTION_SUMMARY in 15 ml tube, vortexed, and incubated on shaker at 150 rpm for 3 hours TCA was
CO:COLLECTION_SUMMARY extracted twice w/ 5ml diethyl ether, then sample was washed with 5 ml
CO:COLLECTION_SUMMARY chloroform, and aqueous phase was recovered Freeze-dried aqueous phase,
CO:COLLECTION_SUMMARY resuspended in 1 ml MeOH and incubated on shaker for 1 hour at 150 rpm,
CO:COLLECTION_SUMMARY centrifuged Supernatant was trasnferred to 2 ml tube, sample washed with
CO:COLLECTION_SUMMARY additional 0.5 ml MeOH and added to 2 ml tube Freeze dried. Note that a method
CO:COLLECTION_SUMMARY blank and 2 recovery standards containing thiamine were included in this
CO:COLLECTION_SUMMARY processing
CO:COLLECTION_PROTOCOL_FILENAME Thiamine_BH_IG032715QTrap.xlsx
CO:SAMPLE_TYPE Cell
#TREATMENT
TR:TREATMENT_SUMMARY BH28 was base degraded thiamine. It did not come from an organism. It was
TR:TREATMENT_SUMMARY suspended thiamine in strong base for an extended period of time. This was done
TR:TREATMENT_SUMMARY to see what types of degraded products would form. BH29-34 were E. coli medium
TR:TREATMENT_SUMMARY samples that were fractionated using a reverse phase column. BH35-36 were media
TR:TREATMENT_SUMMARY samples after E. coli growth where methanol was used for extraction.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY 1. Add 0.5mL of extraction solvent to tube, gently pipet to remove all cells,
SP:SAMPLEPREP_SUMMARY transfer cells to 2mL eppendorf tube. Repeat for a total of 1mL extraction
SP:SAMPLEPREP_SUMMARY solvent + cells in 2mL eppendorf tube. 2. Add 2 small stainless steel grinding
SP:SAMPLEPREP_SUMMARY beads to eppendorf tube 3. Use the GenoGrinder to grind for 3 minutes at 1,250
SP:SAMPLEPREP_SUMMARY rpm. 4. Centrifuge at 14,000xg for 5 minutes. 5. Transfer supernatant to a fresh
SP:SAMPLEPREP_SUMMARY 2mL eppendorf tube. 6. Add 1mL of extraction solvent to tube containing cell
SP:SAMPLEPREP_SUMMARY pellet + beads, and repeat steps 3 and 4. 7. Collect supernatant, and combine
SP:SAMPLEPREP_SUMMARY with supernatant collected in step 5. Total volume of extracted sample will be
SP:SAMPLEPREP_SUMMARY approximately 2mL. 8. Dry down 50uL of extracted sample in 1.5mL eppendorf tube
SP:SAMPLEPREP_SUMMARY for GC-TOF analysis. 9. Store backups in -20 or -80C.
SP:SAMPLEPREP_PROTOCOL_FILENAME SOP_Extraction_of_Yeast_Cells.pdf
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE Reversed phase
CH:INSTRUMENT_NAME Agilent 1290 Infinity
CH:INSTRUMENT_NAME Accurate Mass QTOF
CH:COLUMN_NAME Waters Acquity BEH C18 (100 x 2.1mm, 1.7um)
CH:FLOW_GRADIENT 0% B to 99%B
CH:FLOW_RATE 0.5 mL/min
CH:COLUMN_TEMPERATURE 65 C
CH:SOLVENT_A Water/Acetonitrile 95:5 with 0.1% acetic acid
CH:SOLVENT_B 100% Acetonitrile with 0.1% acetic acid
CH:COLUMN_PRESSURE 1200 bar
CH:INTERNAL_STANDARD CUDA
CH:SAMPLE_INJECTION 3uL
CH:ANALYTICAL_TIME 15 min
CH:TARGET_SAMPLE_TEMPERATURE Autosampler temp 4 C
CH:RANDOMIZATION_ORDER Excel generated
#ANALYSIS
AN:ANALYSIS_TYPE MS
AN:LABORATORY_NAME WCMC Metabolomics Core
#MS
MS:INSTRUMENT_NAME Agilent 6530 QTOF
MS:INSTRUMENT_NAME Accurate Mass QTOF
MS:INSTRUMENT_TYPE QTOF
MS:MS_TYPE ESI
MS:ION_MODE POSITIVE
MS:MS_COMMENTS none
MS:CAPILLARY_VOLTAGE 3500
MS:COLLISION_GAS Nitrogen
MS:DRY_GAS_FLOW 8 L/min
MS:DRY_GAS_TEMP 325 C
MS:FRAGMENT_VOLTAGE 120
MS:FRAGMENTATION_METHOD Auto MS/MS
MS:ION_SOURCE_TEMPERATURE 325 C
MS:ION_SPRAY_VOLTAGE 1000
MS:IONIZATION Pos
MS:PRECURSOR_TYPE Intact Molecule
MS:REAGENT_GAS Nitrogen
MS:SOURCE_TEMPERATURE 325 C
MS:DATAFORMAT .d
MS:DESOLVATION_GAS_FLOW 11 L/min
MS:DESOLVATION_TEMPERATURE 350 C
MS:NEBULIZER 35 psig
MS:OCTPOLE_VOLTAGE 750
MS:RESOLUTION_SETTING extended dynamic range
MS:SCAN_RANGE_MOVERZ 60-1700 Da
MS:SCANNING_CYCLE 2 Hz
MS:SCANNING_RANGE 60-1700 Da
MS:SKIMMER_VOLTAGE 65
MS:MS_RESULTS_FILE ST000400_AN001818_Results.txt UNITS:counts Has m/z:Yes Has RT:Yes RT units:Minutes
#END