| Method |
"21.E. coli
strain BW25113 (
37
) was grown with shaking at 37°C in LB or Chelex-treated (Bio-Rad) A
minimal medium (
38
) containing 0.2% glucose and
40
m
g/ml of all 20
L
-amino acids to an optical
density at 600 nm of 0.5 to 0.6. The cells were
harvested at midlog growth and washed three
times with fresh media containing 1 mM EDTA and
one time with Milli-Q water. The pelleted cells
were dried overnight at 80°C in Teflon tubes,
dissolved in concentrated nitric acid, and the metal
content measured by ICP-MS (PQ ExCell, TJA Solutions, Franklin, MA). Parallel control experiments
were run to determine the background metal contamination in the materials used. All glassware and
plasticware was acid-washed before use.
22. The total cellular concentration of each element was
calculated assuming a maximal cellular volume of 1.8
m
m
3
for cells grown in minimal medium and 3.5
m
m
3
in LB medium (
23
,
31
). Metal concentrations in media
were determined by inductively coupled plasma
atomic emission spectroscopy (Thermo Jarrell Ash
AtomScan 25 ICP-AES) for Mg, Ca, and K and ICP-MS
for all other elements. Because transition metal concentrations in minimal medium were substantially
lower than in LB, ICP-MS measurements of minimal
media samples employed the standard addition
method. This reduced matrix effects." |