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MB Sample ID: SA018750

Local Sample ID:140619dlvsa01_1
Subject ID:SU000416
Subject Type:Cells
Subject Species:Synechococcus elongatus PCC 7942
Taxonomy ID:1140
Species Group:Microorganism

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Subject:

Subject ID:SU000416
Subject Type:Cells
Subject Species:Synechococcus elongatus PCC 7942
Taxonomy ID:1140
Species Group:Microorganism

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
140619dlvsa01_1SA018750FL004345KaiC mutantGenotype
140619dlvsa01_1SA018750FL004345-Time Point

Collection:

Collection ID:CO000410
Collection Summary:Bacteria were grown in a turbidostat/bioreactor at equal cell density (measured by optical density at 750nm), under a 12:12h Light/Dark cycle. After collection samples were immediately placed on ice and then centrifuged at 5000RPM for 10min at ­4 degrees Celsius. After centrifugation supernatant was decanted and cell pellets were immediately frozen in liquid N2.
Collection Protocol Filename:StudyDesign-SpencerDiamond-10814.pdF
Sample Type:Bacterial cells
Collection Time:Samples were collected at T0 (beginning of day) and T4 (4h into day).
Volumeoramount Collected:40ml of sample was collected at each time point
Storage Conditions:Samples were put into a 50mL conical tube containing ice up to the 30ml mark.

Treatment:

Treatment ID:TR000430
Treatment Summary:2: WT bacteria and KaiC mutant The phenotype associated with loss of the core oscillator protein, KaiC, is distinct from that caused by absence of the circadian output transcriptional regulator, RpaA (regulator of phycobilisome-associated A). Untargeted metabolomics analysis and glycogen kinetics suggest that functional KaiC is important for metabolite partitioning in the morning. Additionally, output from the oscillator functions to inhibit RpaA activity in the morning, and kaiC-null strains expressing a mutant KaiC phosphomimetic, KaiC-pST, in which the oscillator is locked in the most active output state, phenocopies a ΔrpaA strain. KaiC-null strains show indications of oxidative pentose phosphate pathway activation as well as increased abundance of primary metabolites. Inhibitory clock output may serve to allow secondary metabolite biosynthesis in the morning, and some metabolites resulting from these processes may feed back to reinforce clock timing.
Treatment Protocol Filename:StudyDesign-SpencerDiamond-10814.pdf

Sample Preparation:

Sampleprep ID:SP000423
Sampleprep Summary:1. Add 0.5mL of extraction solvent to tube, gently pipet to remove all cells, transfer cells to 2mL eppendorf tube. Repeat for a total of 1mL extraction solvent + cells in 2mL eppendorf tube. 2. Add 2 small stainless steel grinding beads to eppendorf tube 3. Use the GenoGrinder to grind for 3 minutes at 1,250 rpm. 4. Centrifuge at 14,000xg for 5 minutes. 5. Transfer supernatant to a fresh 2mL eppendorf tube. 6. Add 1mL of extraction solvent to tube containing cell pellet + beads, and repeat steps 3 and 4. 7. Collect supernatant, and combine with supernatant collected in step 5. Total volume of extracted sample will be approximately 2mL. 8. Dry down 50uL of extracted sample in 1.5mL eppendorf tube for GC-TOF analysis. 9. Store backups in -20 or -80C.
Sampleprep Protocol Filename:SOP_Extraction_of_Yeast_Cells.pdf

Combined analysis:

Analysis ID AN000632
Analysis type MS
Chromatography type GC
Chromatography system Leco Pegasus III GC
Column Restek Rtx-5Sil MS (30 x 0.25mm, 0.25um)
MS Type EI
MS instrument type GC-TOF
MS instrument name Leco Pegasus III GC TOF
Ion Mode POSITIVE
Units counts

Chromatography:

Chromatography ID:CH000457
Methods Filename:SOP_Extraction_of_Yeast_Cells.pdf
Instrument Name:Leco Pegasus III GC
Column Name:Restek Rtx-5Sil MS (30 x 0.25mm, 0.25um)
Column Pressure:7.7 PSI
Column Temperature:50-330C
Flow Rate:1 ml/min
Injection Temperature:50 C ramped to 250 C by 12 C/s
Sample Injection:0.5 uL
Oven Temperature:50°C for 1 min, then ramped at 20°C/min to 330°C, held constant for 5 min
Transferline Temperature:230C
Washing Buffer:Ethyl Acetate
Sample Loop Size:30 m length x 0.25 mm internal diameter
Randomization Order:Excel generated
Chromatography Type:GC

MS:

MS ID:MS000565
Analysis ID:AN000632
Instrument Name:Leco Pegasus III GC TOF
Instrument Type:GC-TOF
MS Type:EI
Ion Mode:POSITIVE
Ion Source Temperature:250 C
Ionization Energy:70 eV
Source Temperature:250 C
Scan Range Moverz:85-500 Da
Scanning Cycle:17 Hz
Scanning Range:85-500 Da
Skimmer Voltage:1850 V
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