Biology Reference
In-Depth Information
Decant and discard the supernatant.
Immediately, add 500 mL quenched yeast suspension, prepared as
described above while the centrifuge is running, on top of each pellet.
Repeat until the entire content of the bioreactor has been harvested.
After the last aliquot of quenched yeast suspension has been centrifuged,
re-suspend each pellet in 200 mL of fresh quenching solution. Aliquot
20 mL into 50-mL Falcon tubes. Keep all components below
20
C
throughout the entire procedure.
Centrifuge again and discard the supernatant.
Store the biomass at
80
C until extraction.
Extraction
Prepare 1.1 L extraction solution: 75:25 ethanol/water with 10 mM
ammonium acetate pH 7.0.
Aliquot 24
20 mL of extraction solution in 50-mL Falcon tubes and
preheat to 78
C.
Pre-warm the quenched samples to
20
C in a cryostat.
Pour 20 mL preheated extraction solution directly from the Falcon tube to
the pellet of the quenched sample and re-suspend immediately using a
25-mL pipette.
Incubate the sample in a water bath for 3 min at 78
C, vortexing
every 60 s.
20
C until centrifugation.
Separate cell debris from metabolite extract by centrifugation at 4000
Incubate the extracts in the cryostat at
g
4
C.
Pool all of the extracts and store at
and
<
80
C as aliquots.
on 100% U-
13
C-labelled glucose
This method describes a convenient bench top cultivation to obtain internal
standard from an anaerobic culture of
E. coli
. Anaerobic conditions are required
to prevent the incorporation of
12
C carbon in metabolites via CO
2
assimilation.
However, it should be noted that the anaerobic conditions significantly reduce the
biomass yield and thus the amount of U-
13
C-labelled metabolites.
Prepare 100 mL of a minimal salt media with 2 g L
1
U-
13
C glucose and
degas with nitrogen.
Grow an anaerobic overnight pre-culture in 5 mL media to an OD
600
of
about 1.
Prepare an anaerobic flask and fill with 95 mL of medium.
Inoculate the anaerobic flask with 5 mL of the pre-culture.
After
2. Anaerobic batch cultivation of
E. coli
6-7 h, when the culture has reached anOD
600
of 0.5, start sampling.
Prepare five glass bottles filled with 10 mL of 60:40 ethanol/water and
preheat to 80
C.
Place a 0.45-
m
m pore size nitrocellulose filter with 90 mm diameter on a
vacuum filter flask and apply a moderate vacuum.
