M9 medium (per liter deionized water): 0.8 g of NH4Cl, 0.5 g of NaCl, 7.5 g of Na2HPO4 · 2H2O, and 3.0 g of KH2PO4. The following components were sterilized separately and then added (per liter [final volume] of medium): 2 ml of 1 M MgSO4, 1 ml of 0.1 M CaCl2, 0.3 ml of 1 mM filter-sterilized thiamine HCl, and 10 ml of a trace element solution containing (per liter) 1 g of FeCl3 · 6H2O, 0.18 g of ZnSO4 · 7H2O, 0.12 g of CuCl2 · 2H2O, 0.12 g of MnSO4 · H2O, and 0.18 g of CoCl2 · 6H2O. Sterilized glucose was added to a final concentration of 2 or 3 g per liter.

Metabolic flux ratio analysis by GC-MS - Samples for gas chromatography (GC)-mass spectrometry (MS) analysis were prepared as described previously [1]. Briefly, aliquots of 13C-labeled batch cultures were withdrawn during the mid-exponential growth phase, which was defined a ... s an OD600 of 0.8 to 1.2 for aerobic cultures and an OD600 of 0.4 to 0.7 for anaerobic cultures. Cell pellets were hydrolyzed in 6 M HCl at 105 °C for 24 h in sealed microtubes. The hydrolysates were dried under a stream of air at around 60°C and then derivatized at 85°C in 30 µl of dimethylformamide (Fluka, Buchs, Switzerland) and 30µl of N-(tert-butyldimethylsilyl)-N-methyl-trifluoroacetamide with 1% (vol/vol) tert-butyldimethylchlorosilane (Fluka) for 60 min. Derivatized amino acids were analyzed with a series 8000 gas chromatograph combined with an MD 800 mass spectrometer (Fisons Instruments, Beverly, Mass.). The GC-MS-derived mass isotope distributions of proteinogenic amino acids were then corrected for naturally occurring isotopes [1]. The corrected mass distributions were related to the in vivo metabolic activities with previously described algebraic equations and statistical data treatment, which quantified nine ratios of fluxes through converging reactions and pathways for the synthesis of five intracellular metabolites [1].
13C-constrained metabolic net flux analysis - Intracellular net carbon fluxes were estimated by using the previously described [2] stoichiometric model.

-----------References------------
[1] Fischer, E., and U. Sauer. 2003. Metabolic flux profiling of Escherichia coli mutants in central carbon metabolism using GC-MS. Eur. J. Biochem. 270: 880–891. http://doi.org/dg3q64
[2] Fischer, E., N. Zamboni, and U. Sauer. 2004. High-throughput metabolic flux analysis based on gas chromatography-mass spectrometry derived 13C constraints. Anal. Biochem. 325:308–316. http://doi.org/c6kx35