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MB Sample ID: SA176607
| Local Sample ID: | TM-1526-1 |
| Subject ID: | SU001982 |
| Subject Type: | Bacteria |
| Subject Species: | Bacteroides thetaiotaomicron |
| Taxonomy ID: | 818 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU001982 |
| Subject Type: | Bacteria |
| Subject Species: | Bacteroides thetaiotaomicron |
| Taxonomy ID: | 818 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| TM-1526-1 | SA176607 | FL021896 | deltaBT1526 | Genotype |
Collection:
| Collection ID: | CO001975 |
| Collection Summary: | B. thetaiotaomicron strains (wild-type and ΔBT1522-BT1526 mutants) were grown overnight in an anaerobic chamber (Coy Laboratories) using an atmosphere of 10% H2, 5% CO2, 85% N2. For liquid growth, Brain heart infusion (BHI) supplemented with hemin and vitamin K3 was used. Cultures were then subjected to subcellular fractionation to obtain total membranes and outer membrane vesicles (OMV) preparation. OMV preparations: OMV were purified by ultracentrifugation of filtered spent media from 150 ml of liquid culture as described previously (1). OMV preparations were resuspended in PBS before lipid analyses. Protein content was quantified using a DC protein assay kit (Bio-Rad). Fractions were aliquoted and stored at -80°C until analyzed. Membrane preparations: Total membrane preparations were performed by cell lysis and ultracentrifugation as previously described (1). Total membranes from 150 ml of liquid culture were resuspended in PBS using a 2-ml glass tissue grinder with a polytetrafluoroethylene (PTFE) pestle (VWR). Protein content was quantified using a DC protein assay kit (Bio-Rad). Fractions were stored aliquoted and stored at -80°C until analyzed. References: 1. Elhenawy W, Debelyy MO, Feldman MF. Preferential packing of acidic glycosidases and proteases into Bacteroides outer membrane vesicles. mBio. 2014;5(2):e00909-14. |
| Sample Type: | Bacterial cells |
Treatment:
| Treatment ID: | TR001994 |
| Treatment Summary: | No treatment displayed. Wild-type and mutant bacteria were grown in BHI supplemented with vitamin K and Hemin |
Sample Preparation:
| Sampleprep ID: | SP001988 |
| Sampleprep Summary: | Total lipids from OMV and TM were extracted based on the Bligh and Dyer chloroform:methanol method (1). Briefly, 2 volumes of methanol, 1 volume of chloroform, and 0.8 volumes of Milli-Q water were added to 1 volume of PBS-resuspended OMV or TM fractions into solvent-resistant glass tubes. Contents were mixed for 1 min by vortexing and 1 volume of chloroform was added to the mixture. Contents were mixed for another minute and tubes were centrifuged for 5 min at 4000 rpm. After centrifugation, bottom phase (organic) was recovered using a glass Pasteur pipette and stored in solvent-sealed vials at -80°C until lipid analysis by LC-MS. References: 1. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959;37(8):911-7. |
Combined analysis:
| Analysis ID | AN003101 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Agilent 6550 |
| Column | Thermo Betasil C18 (100 x 2.1mm,5um) |
| MS Type | ESI |
| MS instrument type | QTOF |
| MS instrument name | Agilent 6550 QTOF |
| Ion Mode | UNSPECIFIED |
| Units | peak area |
Chromatography:
| Chromatography ID: | CH002288 |
| Chromatography Summary: | LC/MS analyses were conducted on an Agilent 6550 A QTOF instrument with an Agilent 1290 HPLC with autosampler, operated by Agilent Masshunter software (Santa Clara, CA USA). Separation of the total lipid extracts was achieved by a Thermo (Waltham MA, USA) 100 × 2.1 mm BETASIL 5 μm C18 column at a flow rate of 300 μl/min at room temperature. The mobile phase contained 5 mM ammonium formate (pH 5.0) both in solvent A, acetonitrile:water (60:40, v/v), and solvent B, isopropanol:acetonitrile (90:10, v/v). A gradient elution in the following manner was applied: 68% A, 0–1.5 min; 68–55% A, 1.5–4 min; 55–48% A, 4–5 min; 48–42% A, 5–8 min; 42–34% A, 8–11 min; 34–30% A, 11–14 min; 30–25% A, 14–18 min; 25–3% A, 18–23 min; 3–0% A, 25–30 min; 0% A, 30–35 min; 68% A, 35–40 min. Both the positive-ion and negative-ion ESI MS scans were acquired in the mass range of 200-2000 Da at a rate of 2 scans/min. |
| Instrument Name: | Agilent 6550 |
| Column Name: | Thermo Betasil C18 (100 x 2.1mm,5um) |
| Column Temperature: | RT |
| Flow Gradient: | 68% A, 0-1.5 min; 68-55% A, 1.5-4 min; 55-48% A, 4-5 min; 48-42% A, 5-8 min; 42-34% A, 8-11 min; 34-30% A, 11-14 min; 30-25% A, 14-18 min; 25-3% A, 18-23 min; 3-0% A, 25-30 min; 0% A, 30-35 min; 68% A, 35-40 min |
| Flow Rate: | 300ul/min |
| Solvent A: | 60% acetonitrile/40% water; 5 mM ammonium formate, pH 5.0 |
| Solvent B: | 90% isopropanol/10% acetonitrile |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS002883 |
| Analysis ID: | AN003101 |
| Instrument Name: | Agilent 6550 QTOF |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| MS Comments: | ESI MS scans were acquired in the mass range of 200-2000 Da at a rate of 2 scans /min. |
| Ion Mode: | UNSPECIFIED |
