Processes 2018, 6,38
composition. Compiling a literature review in conjunction with laboratory-tested protocols with
demonstrated application to metabolic models, all within a single source, serves as a useful
resource for the computational biology community that should facilitate model building transparency
and reproducibility.
2. Culturing Methods
2.1. Strains and Media
E. coli str. K-12 substr. MG1655 was grown in standard M9 minimal salts medium (6 g/L Na 2 HPO 4 ,
3 g/L KH 2 PO 4 , 1 g/L NH 4 Cl, 0.5 g/L NaCl), supplemented with 1 mL/L 1 M MgSO 4 ·7H 2 O and 10 mL/L
trace metals (0.55 g/L CaCl 2 , 0.10 g/L MnCl 2 ·4H 2 O, 0.17 g/L ZnCl 2 , 0.043 g/L CuCl 2 ·2H 2 O, 0.06 g/L
CoCl 2 ·6H 2 O, 0.06 g/L Na 2 MoO 4 ·2H 2 O, 0.06 g/L Fe(NH 4 ) 2 (SO 4 ) 2 ·6H 2 O, 0.20 g/L FeCl 3 ·6H 2 O) [20,21].
Synechococcus 7002 was grown in A+ synthetic seawater medium (18 g/L NaCl, 0.6 g/L KCl, 1 g/L
NaNO 3 , 5 g/L MgSO 4 ·7H 2 O, 0.05 g/L KH 2 PO 4 , 0.27 g/L CaCl 2 , 0.03 g/L Na 2 ethylenediaminetetraacetic
acid, 0.004 g/L FeCl 3 ·6H 2 O, 1 g/L Tris pH 8.2), supplemented with 4 mg/L vitamin B12 and 1 mL/L P1
trace metal mix (34.26 g/L H 3 BO 3 , 4.32 g/L MnCl 2 ·4H 2 O, 0.315 g/L ZnCl 2 , 0.03 g/L MoO 3 , 0.003 g/L
CuSO 4 ·5H 2 O, 0.0122 g/L CoCl 2 ·6H 2 O) [22,23].
A. acidocaldarius str. acidocaldarius DSM446 was grown in Bacillus acidocaldarius medium (BAM)
(1 g/L KH 2 PO 4 , 1.5 g/L (NH 4 ) 2 SO 4 ), supplemented with 0.2 g/L MgSO 4 ·7H 2 O, 0.1 g/L CaCl 2 ·2H 2 O,
and 1 mL/L trace metal mix (10 g/L FeSO 4 ·7H 2 O, 0.1 g/L H 3 BO 3 , 0.15 g/L MnSO 4 ·H 2 O, 0.18 g/L
ZnSO 4 ·7H 2 O, 0.2 g/L CuSO 4 ·5H 2 O, 0.3 g/L Na 2 MoO 4 ·2H 2 O, 0.18 g/L CoCl 2 ·6H 2 O) (modified from
Farrand et al. [24]).
2.2. Culture Conditions
E. coli cultures were grown at 37 ◦ C shaking at 150 rpm. Inoculum cultures were prepared in 8 mL
of M9 + 10 g/L glucose in disposable culture tubes, inoculated with multiple isolated colonies from an
agar plate streaked from a 20% glycerol freezer stock, and grown to OD 600 < 0.6 (exponential phase).
Cells were then centrifuged at 4000 rpm for 10 min and re-suspended to OD 600 ~0.05 in 50 mL of fresh
M9 + 1 g/L glucose in 250-mL baffled shake flasks. Cultures were grown to OD 600 ~0.6 (exponential
phase) and then harvested for analysis (collected in chilled 50-mL polypropylene centrifuge tubes on
ice followed by centrifugation).
Synechococcus 7002 cultures were grown at 38 ◦ C without shaking under continuous light.
Inoculum cultures were prepared in 25 mL of A+ media in 250-mL non-baffled shake flasks, inoculated
with multiple isolated colonies from an agar plate (transferred monthly for propagation), and grown
to OD 730 < 0.5. Cells were then centrifuged at 4000 rpm for 10 min and re-suspended in 25 or 50 mL of
fresh A+ media to an OD 730 ~0.1. Cultures were grown to OD 730 0.4–0.5 and harvested for analysis.
A. acidocaldarius cultures were grown at 60 ◦ C shaking at 200 rpm. Inoculum cultures were
prepared in 50 mL of BAM + 5 g/L glucose in 250-mL baffled shake flasks, inoculated with multiple
isolated colonies from an agar plate streaked from a 20% glycerol freezer stock, and grown to
OD 600 < 0.6. Cells were then centrifuged at 4000 rpm for 10 min and re-suspended to OD 600 ~ 0.1
in 50 mL of fresh BAM + 5 g/L glucose. Cultures were grown to OD 600 ~ 0.6 and then harvested
for analysis.
2.3. Dry Weight Determination
Optical density was correlated to biomass for each organism to determine amount of dry weight
used for macromolecular analyses. Because optical density can fluctuate at high cell concentrations
due to shading effects, samples were diluted to an optical density reading below 0.3 to remain within
the linearity of the spectrophotometer. Biomass to OD 600 correlation for E. coli of 0.5 g/L cell dry
weight per unit OD 600 was obtained from Folsom, Parker, and Carlson [25], which used the same
strain and was performed in the same laboratory using the methods below.
156
composition. Compiling a literature review in conjunction with laboratory-tested protocols with
demonstrated application to metabolic models, all within a single source, serves as a useful
resource for the computational biology community that should facilitate model building transparency
and reproducibility.
2. Culturing Methods
2.1. Strains and Media
E. coli str. K-12 substr. MG1655 was grown in standard M9 minimal salts medium (6 g/L Na 2 HPO 4 ,
3 g/L KH 2 PO 4 , 1 g/L NH 4 Cl, 0.5 g/L NaCl), supplemented with 1 mL/L 1 M MgSO 4 ·7H 2 O and 10 mL/L
trace metals (0.55 g/L CaCl 2 , 0.10 g/L MnCl 2 ·4H 2 O, 0.17 g/L ZnCl 2 , 0.043 g/L CuCl 2 ·2H 2 O, 0.06 g/L
CoCl 2 ·6H 2 O, 0.06 g/L Na 2 MoO 4 ·2H 2 O, 0.06 g/L Fe(NH 4 ) 2 (SO 4 ) 2 ·6H 2 O, 0.20 g/L FeCl 3 ·6H 2 O) [20,21].
Synechococcus 7002 was grown in A+ synthetic seawater medium (18 g/L NaCl, 0.6 g/L KCl, 1 g/L
NaNO 3 , 5 g/L MgSO 4 ·7H 2 O, 0.05 g/L KH 2 PO 4 , 0.27 g/L CaCl 2 , 0.03 g/L Na 2 ethylenediaminetetraacetic
acid, 0.004 g/L FeCl 3 ·6H 2 O, 1 g/L Tris pH 8.2), supplemented with 4 mg/L vitamin B12 and 1 mL/L P1
trace metal mix (34.26 g/L H 3 BO 3 , 4.32 g/L MnCl 2 ·4H 2 O, 0.315 g/L ZnCl 2 , 0.03 g/L MoO 3 , 0.003 g/L
CuSO 4 ·5H 2 O, 0.0122 g/L CoCl 2 ·6H 2 O) [22,23].
A. acidocaldarius str. acidocaldarius DSM446 was grown in Bacillus acidocaldarius medium (BAM)
(1 g/L KH 2 PO 4 , 1.5 g/L (NH 4 ) 2 SO 4 ), supplemented with 0.2 g/L MgSO 4 ·7H 2 O, 0.1 g/L CaCl 2 ·2H 2 O,
and 1 mL/L trace metal mix (10 g/L FeSO 4 ·7H 2 O, 0.1 g/L H 3 BO 3 , 0.15 g/L MnSO 4 ·H 2 O, 0.18 g/L
ZnSO 4 ·7H 2 O, 0.2 g/L CuSO 4 ·5H 2 O, 0.3 g/L Na 2 MoO 4 ·2H 2 O, 0.18 g/L CoCl 2 ·6H 2 O) (modified from
Farrand et al. [24]).
2.2. Culture Conditions
E. coli cultures were grown at 37 ◦ C shaking at 150 rpm. Inoculum cultures were prepared in 8 mL
of M9 + 10 g/L glucose in disposable culture tubes, inoculated with multiple isolated colonies from an
agar plate streaked from a 20% glycerol freezer stock, and grown to OD 600 < 0.6 (exponential phase).
Cells were then centrifuged at 4000 rpm for 10 min and re-suspended to OD 600 ~0.05 in 50 mL of fresh
M9 + 1 g/L glucose in 250-mL baffled shake flasks. Cultures were grown to OD 600 ~0.6 (exponential
phase) and then harvested for analysis (collected in chilled 50-mL polypropylene centrifuge tubes on
ice followed by centrifugation).
Synechococcus 7002 cultures were grown at 38 ◦ C without shaking under continuous light.
Inoculum cultures were prepared in 25 mL of A+ media in 250-mL non-baffled shake flasks, inoculated
with multiple isolated colonies from an agar plate (transferred monthly for propagation), and grown
to OD 730 < 0.5. Cells were then centrifuged at 4000 rpm for 10 min and re-suspended in 25 or 50 mL of
fresh A+ media to an OD 730 ~0.1. Cultures were grown to OD 730 0.4–0.5 and harvested for analysis.
A. acidocaldarius cultures were grown at 60 ◦ C shaking at 200 rpm. Inoculum cultures were
prepared in 50 mL of BAM + 5 g/L glucose in 250-mL baffled shake flasks, inoculated with multiple
isolated colonies from an agar plate streaked from a 20% glycerol freezer stock, and grown to
OD 600 < 0.6. Cells were then centrifuged at 4000 rpm for 10 min and re-suspended to OD 600 ~ 0.1
in 50 mL of fresh BAM + 5 g/L glucose. Cultures were grown to OD 600 ~ 0.6 and then harvested
for analysis.
2.3. Dry Weight Determination
Optical density was correlated to biomass for each organism to determine amount of dry weight
used for macromolecular analyses. Because optical density can fluctuate at high cell concentrations
due to shading effects, samples were diluted to an optical density reading below 0.3 to remain within
the linearity of the spectrophotometer. Biomass to OD 600 correlation for E. coli of 0.5 g/L cell dry
weight per unit OD 600 was obtained from Folsom, Parker, and Carlson [25], which used the same
strain and was performed in the same laboratory using the methods below.
156