Measurement Of Escherichia Coli TUQ2 Central Carbon Metabolic Flux Distribution Under Anaerobic Condition Using ¹³C Labeling Technique

This paper adopts the ¹³C labeling technique combined with Gas Chromalography-Mass Spectrum (GC-MS) measurement technique as quantitive analysis method to identify the central metabolic flux distributions of Escherichia coli TUQ2 under anaerobic enviroment. This project will provide a more accurate bird's view about metabolic flux distributions under ptsG gene knockout condition and will help to locate more protential gene manipulation point for strain production improvement of succinic acid. Due to the limited time, unavailability of equipments and chemicals, this paper only covers preliminary experiments to figure out the procedures and culture parameters of the prophase GC-MS chemostat experiment. Problems such as substrates labeling strategy, GC-MS spectrum analysis, etc. are theoretically analyzed and settled properly.Considering the complexisity of determinating substrate labeling stragety, the selection of labeling pattern in this paper is qualitively decided under the guidance of combined optimization of high GC-MS spectrum resolution and strong signal intensity.The GC-MS samples are prepared from the intermediate metabolite hydrolyzed residues. The intermediate metabolites are derived from the chemostat E. coli culture, after adding ¹³C labeling substrate and waiting for 3 resident times. FiatFlux, which is an intuitive tool for quantitative investigations of intracellular metabolism by users that are not familiar with numerical methods or isotopic tracer experiments. The aim of this open source software is to enable non-specialists to adapt the software to their specific scientific interests, including other ¹³C-substrates, labeling mixtures, and organisms.The calculation of the central metabolic flux distribution in this paper can be devided into four steps. First of all, a model which is suitable for Escherichia coli TUQ2 is founded,and a correction could be made with MATLAB if needed. Secondly, manually assign TBDMS-derivatized proteinogenic amino acids analyzed by GC-MS,and choose a proper assignment.Thirdly,determin the extracellular flux by HPLC and the activity and reversibility of all reactions in the reaction network must be considered during the calculation process.Finally,use least square estimation F between simulation calculation and experiment data as the optimization objective to solve the central metabolic flux distribution of Escherichia coli TUQ2 under anaerobic enviroment.