#METABOLOMICS WORKBENCH michaelsa93_20160513_094817_mwtab.txt DATATRACK_ID:629 STUDY_ID:ST000400 ANALYSIS_ID:AN001819 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 6550 QTOF MS:INSTRUMENT_NAME Accurate Mass QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS none MS:CAPILLARY_VOLTAGE 3500 MS:COLLISION_GAS Nitrogen MS:DRY_GAS_FLOW 13 L/min MS:DRY_GAS_TEMP 200 C MS:FRAGMENT_VOLTAGE 175 MS:FRAGMENTATION_METHOD Auto MS/MS MS:ION_SOURCE_TEMPERATURE 325 C MS:ION_SPRAY_VOLTAGE 1000 MS:IONIZATION Neg MS:DATAFORMAT .d MS:DESOLVATION_GAS_FLOW 11 L/min MS:DESOLVATION_TEMPERATURE 350 C MS:NEBULIZER 35 psig MS:OCTPOLE_VOLTAGE 750 MS:SCAN_RANGE_MOVERZ 60-1700 Da MS:SCANNING_CYCLE 2Hz MS:SCANNING_RANGE 60-1700 MS:SKIMMER_VOLTAGE 65 MS:MS_RESULTS_FILE ST000400_AN001819_Results.txt UNITS:counts Has m/z:Yes Has RT:Yes RT units:Minutes #END