Metabolic flux analysis using isotope distribution together with gene and protein expression analyses

为了建立大肠杆菌主要代谢途径的定量模型，我们培养了E.coliK12和W3110，以及庆应大学培育的E.coliBW25113及其几个单基因敲除突变体(zwf^-、pykF^-、GND^-、ppc^-、pck^-、pgi^-等)。以批处理和连续模式。我们还培育了几个基因敲除突变体(pflA、B、C、D-、PTA-、PPC-、Adha-等)。在微氧条件下生产乳酸盐。在此基础上进行代谢通量分析，利用RT-PCR技术检测酶活性、细胞内代谢物浓度和基因表达，定量了解代谢调控规律，为建模提供依据。

In order to develop a quantitative model for the main metabolic pathway of Escherichia coli, we cultivated E.coliK12 and W3110, as well as E.coli BW25113 developed in Keio University, and its several single-gene-knockout mutants (zwf^-, pykF^-, gnd^-, ppc^-, pck^-, pgi^- etc.) in batch and continuous modes. We also cultivated several gene knockout mutants (pflA, B, C, D-, pta-, ppc-, adhA- etc.) in microaerobic condition for lactate production. Then we made the metabolic flux analysis based on ^<13>C labeling experiments, measured enzyme activities and the intracellular metabolite concentrations as well as gene expressions using RT-PCR to understand the metabolic regulation quantitatively for the modeling.