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MB Sample ID: SA183174

Local Sample ID:	20210227-3-50-2
Subject ID:	SU002017
Subject Type:	Bacteria
Subject Species:	Escherichia coli
Taxonomy ID:	562

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Subject:

Subject ID:	SU002017
Subject Type:	Bacteria
Subject Species:	Escherichia coli
Taxonomy ID:	562

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
20210227-3-50-2	SA183174	FL022312	0.25%	Concentration of MgSO4(w/v)

Collection:

Collection ID:	CO002010
Collection Summary:	After 72 hours of culture, the planktonic cells were isolated, and the biofilms were washed with PBS solvent for three times. Then, planktonic cells and biofilm solutions were centrifuged at 2000 rpm and 4°C for 15 minutes, to collect the strain pellets.
Sample Type:	Biofilm

Treatment:

Treatment ID:	TR002029
Treatment Summary:	Colony-forming antigen (CFA) medium (1% casamino acids, 0.15% yeast extract, 0.005% magnesium sulfate, and 0.0005% manganese chloride) was used to form biofilms. The E. coli strain was incubated in LB-agar plate for 12 hours, one colony was isolated to LB broth for further 4 hours incubation, then diluted the solution into CFA medium at a ratio of 1:1000 and the cultures were incubated for another 72 h at 30°C to form mature biofilms. To test the influence of MgSO4 on biofilm formation, the purified compound of MgSO4 was added to the CFA medium at different concentrations for the preparation of treated culture medium; The left cultural procedures were kept as the same as the protocol listed above.

Treatment ID:

TR002029

Treatment Summary:

Colony-forming antigen (CFA) medium (1% casamino acids, 0.15% yeast extract, 0.005% magnesium sulfate, and 0.0005% manganese chloride) was used to form biofilms. The E. coli strain was incubated in LB-agar plate for 12 hours, one colony was isolated to LB broth for further 4 hours incubation, then diluted the solution into CFA medium at a ratio of 1:1000 and the cultures were incubated for another 72 h at 30°C to form mature biofilms. To test the influence of MgSO4 on biofilm formation, the purified compound of MgSO4 was added to the CFA medium at different concentrations for the preparation of treated culture medium; The left cultural procedures were kept as the same as the protocol listed above.

Sample Preparation:

Sampleprep ID:	SP002023
Sampleprep Summary:	Each pellet sample was mixed with 2 mL of 80% ice-cold methanol, and the mixed solution was homogenized on ice for 2 minutes and repeated the same procedure for three times. Next, the collected samples were centrifuged at 12000 rpm and 4°C for 15 minutes, to collect the supernatants. The supernatants were further mixed with 800 μL of ice-cold acetonitrile for 20 minutes before further centrifuge at 12000 rpm and 4°C for 15 minutes. At last, the isolated supernatants were gone through a 0.22 μm filter membrane (nylon) before the lyophilization. The lyophilized samples were stored at -80°C for metabolomics analysis.

Sampleprep ID:

SP002023

Sampleprep Summary:

Each pellet sample was mixed with 2 mL of 80% ice-cold methanol, and the mixed solution was homogenized on ice for 2 minutes and repeated the same procedure for three times. Next, the collected samples were centrifuged at 12000 rpm and 4°C for 15 minutes, to collect the supernatants. The supernatants were further mixed with 800 μL of ice-cold acetonitrile for 20 minutes before further centrifuge at 12000 rpm and 4°C for 15 minutes. At last, the isolated supernatants were gone through a 0.22 μm filter membrane (nylon) before the lyophilization. The lyophilized samples were stored at -80°C for metabolomics analysis.

Combined analysis:

Analysis ID	AN003153	AN003154
Analysis type	MS	MS
Chromatography type	Reversed phase	Reversed phase
Chromatography system	Agilent 1290 Infinity	Agilent 1290 Infinity
Column	Waters Acquity BEH HSS T3 (100 x 2.1mm,1.8um)	Waters Acquity BEH HSS T3 (100 x 2.1mm,1.8um)
MS Type	ESI	ESI
MS instrument type	Triple quadrupole	Triple quadrupole
MS instrument name	Agilent 6495 QQQ	Agilent 6495 QQQ
Ion Mode	NEGATIVE	POSITIVE
Units	-	-

Chromatography:

Chromatography ID:	CH002332
Chromatography Summary:	The chromatographic separation of biological samples was implemented on a Waters ACQUITY UPLC HSS T3 column (2.1×100 mm, 1.8 μm) with an optimized gradient-elution program (mobile phase A and B were 0.1% formic acid in water and acetonitrile (v/v)): 0-2 minutes, 98% A; 2-10 minutes, 98%-65% A; 10-12 minutes, 65%-20% A; 12-14 minutes, 20%-2% A; 14-30 minutes, 2% A. The column temperature was at 40°C and the flow rate was set at 0.3 mL/ minutes.
Instrument Name:	Agilent 1290 Infinity
Column Name:	Waters Acquity BEH HSS T3 (100 x 2.1mm,1.8um)
Column Temperature:	40
Flow Gradient:	0-2 minutes, 98% A; 2-10 minutes, 98%-65% A; 10-12 minutes, 65%-20% A; 12-14 minutes, 20%-2% A; 14-30 minutes, 2% A
Flow Rate:	0.3 mL/ min
Solvent A:	100% water; 0.1% formic acid
Solvent B:	100% acetonitrile; 0.1% formic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS002933
Analysis ID:	AN003153
Instrument Name:	Agilent 6495 QQQ
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	Agilent MassHunter Workstation Data Acquisition Agilent MassHunter QualitativeAnalysis B.07.00 Agilent MassHunter Quantitative Analysis (for QQQ)
Ion Mode:	NEGATIVE

MS ID:	MS002934
Analysis ID:	AN003154
Instrument Name:	Agilent 6495 QQQ
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	Agilent MassHunter Workstation Data Acquisition Agilent MassHunter QualitativeAnalysis B.07.00 Agilent MassHunter Quantitative Analysis (for QQQ)
Ion Mode:	POSITIVE