Biology Reference
In-Depth Information
and interpretation (van der Werf
et al.
, 2007b). These reporting standards are even
more relevant for metabolomics than for other 'omics' technologies because intra-
cellular metabolite concentrations are very sensitive to the precise physiological
conditions and the growth phase of the culture. Here, we describe the standard cul-
tivation protocol for quantitative comparison of steady-state metabolite concentra-
tions in different strains and carbon sources that has been tested in several
basysbio.eu
)
.
2.1
Medium
The M9 minimal medium used for cultivation of
E. coli
is composed per litre of
200 mL of heat-sterilized base salt solution containing: 7.5 g L
1
(NH
4
)
2
SO
4
,
2.5 g L
1
NaCl, 37.6 g L
1
Na
2
HPO
4
2H
2
O and 15.0 g L
1
KH
2
PO
4
;
700 mL heat-sterilized double-distilled water;
1 mL of heat-sterilized 1 M MgSO
4
;
1 mL of heat-sterilized 0.1 M CaCl
2
;
0.6 mL of filter-sterilized 0.1 M FeCl
3
6H
2
O;
2 mL of filter-sterilized 1 mM thiamine HCl (add only prior to growth
experiment);
10 mL of a filter-sterilized trace element solution containing: 0.18 g L
1
ZnSO
4
7H
2
O, 0.12 g L
1
CuCl
2
2H
2
O, 0.12 g L
1
MnSO
4
H
2
O and 0.18 g L
1
6H
2
O;
25 mL of the tested carbon source solution in water (heat or filter sterilized);
Use heat-sterilized double-distilled water to fill up to 1 L.
CoCl
2
The M9 minimal medium used for cultivation of
Bacillus subtilis
is composed per
litre of
200 mL of heat-sterilized base salt solution containing: 42.5 g L
1
Na
2
HPO
4
2H
2
O, 15 g L
1
KH
2
PO
4
,5gL
1
NH
4
Cl, 2.5 g L
1
NaCl;
700 mL heat-sterilized double-distilled water;
1 mL heat-sterilized 0.1 M CaCl
2
;
1 mL heat-sterilized 1 M MgSO
4
;
1 mL filter-sterilized 50 mM FeCl
3
6H
2
O
þ
100 mM citric acid;
10 mL filter-sterilized trace element solution containing: 0.17 g L
1
ZnCl
2
,
0.1 g L
1
MnCl
2
4H
2
O, 0.06 g L
1
CoCl
2
6H
2
O, 0.06 g L
1
Na
2
MoO
4
2H
2
O
and 0.043 g L
1
CuCl
2
2H
2
O;
25 mL of the tested carbon source solution in water (heat or filter sterilized);
Use heat-sterilized double-distilled water to fill up to 1 L.
Note that the formation of precipitate after mixing all compounds is normal; there-
fore, shake the bottle well before withdrawing medium.
