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MB Sample ID: SA253105
Local Sample ID: | P1-C05 |
Subject ID: | SU002615 |
Subject Type: | Bacteria |
Subject Species: | Eggerthella lenta |
Taxonomy ID: | 84112 |
Genotype Strain: | DSM 2243, Valencia, AB8n2 |
Select appropriate tab below to view additional metadata details:
Subject:
Subject ID: | SU002615 |
Subject Type: | Bacteria |
Subject Species: | Eggerthella lenta |
Taxonomy ID: | 84112 |
Genotype Strain: | DSM 2243, Valencia, AB8n2 |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
P1-C05 | SA253105 | FL032304 | 2243 | Strain |
P1-C05 | SA253105 | FL032304 | 10 | AcetateConc |
P1-C05 | SA253105 | FL032304 | TP4 | TimePoint |
P1-C05 | SA253105 | FL032304 | 28.5 | Time |
Collection:
Collection ID: | CO002608 |
Collection Summary: | Time course experiments were conducted in tubes in the anaerobic chamber in a 37°C incubator. For all metabolomics experiments, three independent culture replicates were included for each condition, with an equal number of uninoculated control tubes. Starter cultures and inocula were prepared as described above for growth assays. 5mLs of defined media was added to VWR glass culture tubes (53283-800) with screw caps. The PBS-washed inoculum was added to culture tubes to obtain an approximate starting OD600 of 0.001. A preliminary growth assay was conducted to define time points spanning the exponential growth phase in the tested conditions. At each time point, OD600 measurements of all inoculated tubes were first measured using a Hach DR1900 spectrophotometer, with a paired control tube to normalize for the background. 100 μL from each tube were then transferred into a 96-well microplate, which was sealed and removed from the anaerobic chamber. Plates were centrifuged at 1,928 rcf at 4°C for 8 minutes, after which supernatants were collected into fresh polypropylene tubes or plates, sealed, and flash-frozen in liquid nitrogen. |
Sample Type: | Bacterial culture supernatant |
Collection Frequency: | at time points specified in study design table over 64 hours (full growth phase) |
Storage Conditions: | -80℃ |
Treatment:
Treatment ID: | TR002627 |
Treatment Summary: | For growth and metabolomics experiments, glycerol stocks of the 3 E. lenta strains were first streaked on BHI+ agar plates and incubated at 37°C for 2-3 days. Individual colonies were inoculated into 3-4 mL liquid BHI+ and incubated at 37°C for 40-48 hours, or until approximately early stationary phase. Culture optical density (600 nm wavelength absorbance, OD600) was measured using a Hach DR1900 spectrophotometer. 1 mL samples of BHI starter cultures were then centrifuged at 1,568 rcf for 4 minutes in a microcentrifuge (ThermoScientific mySpin 12) in the anaerobic chamber and resuspended in 1 mL sterile phosphate-buffered saline (PBS). The resulting suspension was vortexed and diluted to an approximate OD600 of 0.1, and used as inoculum into defined experimental conditions. Varying media conditions were prepared separately and all allowed to fully reduce in the anaerobic chamber prior to inoculation. |
Sample Preparation:
Sampleprep ID: | SP002621 |
Sampleprep Summary: | Samples (20 μL) were first mixed with an internal standard solution (30 μL; 1 mM phenylpropionate-d9) in a V-bottomed, poly(propylene), 96-well plate, and extracted by mixing with 3 sample volumes of extraction solution (75% acetonitrile:25% methanol). The plate was covered with a lid and centrifuged at 5,000 rcf for 15 min at 4 °C. Supernatant was collected for derivatization before subjecting to LC–MS analysis. Samples were processed using a derivatization method targeting compounds containing a free carboxylic acid. Extracted samples were mixed with 3-nitrophenylhydrazine (NPH; 200 mM in 50% acetonitrile) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (120 mM in 6% pyridine) at a 2:1:1 ratio. The plate was sealed with a plastic sealing mat (Thermo Fisher Scientific, #AB-0566) and incubated at 40 °C, 600 rpm in a thermomixer for 60 min to derivatize the carboxylate-containing compounds. The reaction mixture was quenched with 0.02% formic acid in 10% acetonitrile:water before LC–MS. |
Combined analysis:
Analysis ID | AN004142 |
---|---|
Analysis type | MS |
Chromatography type | Unspecified |
Chromatography system | Agilent 1290 Infinity II |
Column | Waters ACQUITY UPLC CSH C18 (100 x 2.1mm,1.7um) |
MS Type | ESI |
MS instrument type | QTOF |
MS instrument name | Agilent 6545 QTOF |
Ion Mode | NEGATIVE |
Units | mM |
Chromatography:
Chromatography ID: | CH003067 |
Chromatography Summary: | Samples were injected via refrigerated autosampler into mobile phase and chromatographically separated by an Agilent 1290 Infinity II UPLC and detected using an Agilent 6545XT Q-TOF (quadrupole time of flight) mass spectrometer equipped with a dual jet stream electrospray ionization source, operating under extended dynamic range (1,700 m/z). Chromatographic separation was performed using an ACQUITY Bridged Ethylene Hybrid (BEH) C18 column 2.1 x 100 mm, 1.7-micron particle size, (Waters Corp. Milford, MA), using chromatographic conditions published elsewhere (Supplementary Table S26 of https://doi.org/10.1038/s41564-022-01109-9). |
Instrument Name: | Agilent 1290 Infinity II |
Column Name: | Waters ACQUITY UPLC CSH C18 (100 x 2.1mm,1.7um) |
Column Temperature: | 50 |
Flow Gradient: | See |
Flow Rate: | 300 μL per minute |
Solvent A: | 0.1% formic acid in water |
Solvent B: | 0.1% formic acid in methanol |
Chromatography Type: | Unspecified |
MS:
MS ID: | MS003889 |
Analysis ID: | AN004142 |
Instrument Name: | Agilent 6545 QTOF |
Instrument Type: | QTOF |
MS Type: | ESI |
MS Comments: | Samples were injected via refrigerated autosampler into mobile phase and chromatographically separated by an Agilent 1290 Infinity II UPLC and detected using an Agilent 6545XT Q-TOF (quadrupole time of flight) mass spectrometer equipped with a dual jet stream electrospray ionization source, operating under extended dynamic range (1,700 m/z). MS1 spectra were collected in centroid mode, and peak assignments in samples were made based on comparisons of retention times and accurate masses from authentic standards using MassHunter Quantitative Analysis v.10.0 software from Agilent Technologies. Acetate was quantified from calibration curves constructed with acetate-d4 as a standard using isotope-dilution MS with phenylpropionate-d9 as the internal standard. Calibration curves were performed in a modified base form of EDM1 lacking amino acids and other carboxylic acids. A background level of 1.05mM of acetate was subtracted to obtain the final quantities. |
Ion Mode: | NEGATIVE |