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MB Sample ID: SA210085
Local Sample ID: | codY 6h_3 |
Subject ID: | SU002273 |
Subject Type: | Bacteria |
Subject Species: | Staphylococcus aureus |
Taxonomy ID: | 1280 |
Gender: | Not applicable |
Species Group: | Bacteria |
Select appropriate tab below to view additional metadata details:
Subject:
Subject ID: | SU002273 |
Subject Type: | Bacteria |
Subject Species: | Staphylococcus aureus |
Taxonomy ID: | 1280 |
Gender: | Not applicable |
Species Group: | Bacteria |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
codY 6h_3 | SA210085 | FL025636 | ccpA | Genotype |
Collection:
Collection ID: | CO002266 |
Collection Summary: | Strains were inoculated in 3 ml of TSB containing 14 mM glucose in glass tubes (Fisher Scientific, cat# 14-961-29) for 16 h and adjusted to an OD600 of 0.05 in 50 ml TSB containing 14 mM glucose in 500 ml flasks and grown at 250 rpm, 37oC for 3 h or 6 h. Culture volumes corresponding to OD600 of 10 optical units were harvested and rapidly filtered through a membrane (0.45 μm, Millipore Cat# MIHAWG100) using Microfil® Filtration Funnels (Millipore, Cat# HAWG047S6) and EZ-FITTM Manifold Base, 6 Place Stainless Steel (Millipore, Cat# EZFITBASE6). The cells on the membrane were washed twice with 5 ml of 0.6% saline and then immediately quenched in 5 ml of ice-cold 60% ethanol containing Br-ATP (final concentration 2 μM) as internal control. The cells were mechanically disrupted using 0.1 mm diameter Glass beads (biospec, Cat# 11079101) and bead homogenizer for 30 s at 6,800 rpm, 3 cycles, 10 sec pause, 4oC(Precellys Evolution, bertin technologies) and centrifuged at 13,000 rpm, 10 min at 4oC to remove cell debris. The supernatant containing intracellular metabolites were separated, and 3 ml was added to 17 ml DW, frozen, lyophilized using FreeZone 4.5 Liter (labconco, Catalog #: 720401050), , and stored in a ultra-low temperature freezer set at -80°C until further analysis. |
Sample Type: | Bacterial cells |
Storage Conditions: | -20℃ |
Treatment:
Treatment ID: | TR002285 |
Treatment Summary: | Staphylococcus aureus UAMS-1 wild-type strain (WT) Staphylococcus aureus UAMS-1 codY::ermC mutant (codY) Staphylococcus aureus UAMS-1 ccpA::tetM mutant (ccpA) Staphylococcus aureus UAMS-1 codY::ermC ccpA::tetM mutant (codY ccpA) |
Sample Preparation:
Sampleprep ID: | SP002279 |
Sampleprep Summary: | Strains were inoculated in 3 ml of TSB containing 14 mM glucose in glass tubes (Fisher Scientific, cat# 14-961-29) for 16 h and adjusted to an OD600 of 0.05 in 50 ml TSB containing 14 mM glucose in 500 ml flasks and grown at 250 rpm, 37oC for 3 h or 6 h. Culture volumes corresponding to OD600 of 10 optical units were harvested and rapidly filtered through a membrane (0.45 μm, Millipore Cat# MIHAWG100) using Microfil® Filtration Funnels (Millipore, Cat# HAWG047S6) and EZ-FITTM Manifold Base, 6 Place Stainless Steel (Millipore, Cat# EZFITBASE6). The cells on the membrane were washed twice with 5 ml of 0.6% saline and then immediately quenched in 5 ml of ice-cold 60% ethanol containing Br-ATP (final concentration 2 μM) as internal control. The cells were mechanically disrupted using 0.1 mm diameter Glass beads (biospec, Cat# 11079101) and bead homogenizer for 30 s at 6,800 rpm, 3 cycles, 10 sec pause, 4oC(Precellys Evolution, bertin technologies) and centrifuged at 13,000 rpm, 10 min at 4oC to remove cell debris. The supernatant containing intracellular metabolites were separated, and 3 ml was added to 17 ml DW, frozen, lyophilized using FreeZone 4.5 Liter (labconco, Catalog #: 720401050), , and stored in a ultra-low temperature freezer set at -80°C until further analysis. |
Processing Storage Conditions: | On ice |
Extract Storage: | -80℃ |
Combined analysis:
Analysis ID | AN003581 |
---|---|
Analysis type | MS |
Chromatography type | HILIC |
Chromatography system | Waters Acquity I-Class |
Column | Waters Acquity BEH Amide (150 x 2.1mm,1.7um) |
MS Type | ESI |
MS instrument type | Triple quadrupole |
MS instrument name | ABI Sciex 6500+ QTrap |
Ion Mode | UNSPECIFIED |
Units | CPS |
Chromatography:
Chromatography ID: | CH002648 |
Chromatography Summary: | Triple-quadrupole-ion trap hybrid mass spectrometer viz., QTRAP6500+ (Sciex, USA) connected with ultra-performance liquid chromatography I-class (UPLC) system procured from Waters, USA was used for the metabolite analysis. The chromatographic separation was performed by liquid chromatography using XBridge Amide (150 × 2.1mm ID; 3.5µm particle size, Waters, USA) analytical column and a binary solvent system with a flow rate of 0.4 ml/min. A guard XBridge Amide column (20 × 2.1mm ID; 3.5µm particle size, Waters, USA) was connected in front of analytical column. Mobile phase A was composed of 10 mM ammonium acetate, 10mM ammonium hydroxide containing 5% acetonitrile in LC-MS grade water; mobile phase B was 100% LC-MS grade acetonitrile. |
Instrument Name: | Waters Acquity I-Class |
Column Name: | Waters Acquity BEH Amide (150 x 2.1mm,1.7um) |
Flow Rate: | 0.4 ml/min |
Solvent A: | 5% acetonitrile/95% water; 10 mM ammonium acetate; 10 mM ammonium hydroxide |
Solvent B: | 100% acetonitrile |
Chromatography Type: | HILIC |
MS:
MS ID: | MS003338 |
Analysis ID: | AN003581 |
Instrument Name: | ABI Sciex 6500+ QTrap |
Instrument Type: | Triple quadrupole |
MS Type: | ESI |
MS Comments: | QTRAP6500+ operated in polarity switching mode was used for targeted quantitation of amino acids through Multiple Reaction Monitoring (MRM) process. Electrospray ionization (ESI) parameters were optimized are as follows: electrospray ion voltage of -4200V and 5500V in negative and positive mode respectively, source temperature of 400°C, curtain gas of 35, and gas 1 and 2 of 40 and 40 psi, respectively. Compound specific parameters were optimized for each compound using manual tuning. These parameters are declustering potential (DP) were 65V and -60V in positive and negative mode respectively, entrance potential (EP) was set at 10V and -10V in positive and negative mode respectively and collision cell exit potential (CXP) was maintained at 10V and -10V in positive and negative mode respectively. MRM data was analysed using Multiquant software. |
Ion Mode: | UNSPECIFIED |