PubMed · 4891998
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S Makover. 1968. A preferred origin for the replication of lambda DNA.. https://doi.org/10.1101/sqb.1968.033.01.068
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A widely applicable cultivation strategy, which reduces the costs of expensive isotopes, is designed for maximal (98-100%) incorporation of [13C] and [15N] into labelled recombinant protein expressed in Escherichia coli, allowing better assignment of the resonances for NMR studies. Isotope labelling of the culture was performed throughout the complete process, starting from preculture. Sufficient biomass is first generated in a batch phase. Upon consumption of glucose, identified by a sharp drop of on-line monitored oxygen consumption, expression is induced and cultivation is continued under glucose-limited conditions as fed-batch process. Thereby a quantitative utilisation of the most expensive component [13C]-glucose is achieved, while the approximate amount of the [15N]-ammonium chloride to be incorporated is calculated from the scheduled biomass. The usefulness of the strategy is demonstrated with production of uniformly [13C/15N]-labelled tryparedoxin of Crithidia fasciculata. Ideal isotope incorporation and product quality is documented by MALDI-TOF mass spectrometry and two- and three-dimensional NMR spectra.
13C-constrained flux balancing analysis based on gas chromatography-mass spectrometry data is presented here as a simple and robust method for the estimation of intracellular carbon fluxes. In this approach, the underdetermined system of metabolite balances deduced from stoichiometric relations and measured extracellular rates is complemented with 13C constraints from metabolic flux ratio analysis. Fluxes in central carbon metabolism of exponentially growing Escherichia coli were estimated by 13C-constrained flux balancing from three different 13C-labeled glucose experiments. The best resolution of the network was achieved using 13C constraints derived from [U-13C]glucose and [1-13C]glucose experiments. The corresponding flux estimate was in excellent agreement with a solution that was independently obtained with a comprehensive isotopomer model. This new methodology was also demonstrated to faithfully capture the intracellular flux distribution in E. coli shake flasks and 1-ml deep-well microtiter plates. Due to its simplicity, speed, and robustness, 13C-constrained metabolic flux balancing is promising for routine and high-throughput analysis on a miniaturized scale.
Phospholipids are major building blocks for biological membranes. In addition, metabolites derived from their degradation are important signals in major cellular events, such as proliferation and apoptosis. The concept of lipid signaling in cells is derived mainly from the measurement of change in the concentration of lipid molecules. However, these changes in concentration are only a small part of the underlying metabolic change induced by a perturbation in the cell. In contrast, metabolic kinetic studies documenting product-precursor relationships and turnover rates are useful in elucidating the responsible mechanisms. Historically, metabolic studies of phospholipids in cells have been carried out with pulse or pulse-chase methods using radioactive isotopes. While these studies provide valuable information, their scope is restricted by inherent limitations. In this paper we describe a method using [1,2,3,4-13C(4)]palmitate as the tracer for studying the metabolic kinetics of the molecular species of diacylglycerol, ceramide, phosphatidylcholine, and sphingomyelin. After growing cells in the presence of labeled palmitate complexed to serum albumin, the lipids are extracted and separated into lipid classes. After enzymatic hydrolysis, diacylglyerols and ceramides as bis-trimethylsilyl derivatives are determined quantitatively with capillary column gas chromatography. Internal standards for each lipid class are used in the procedure. In addition, the isotopic enrichments of the lipid molecular species are determined with gas chromatograph-mass spectrometry. We applied this method to the study of HL60 cells. Different turnover rates were found for various molecular species. In addition, the sn-1 and sn-2 acyl groups appear to be synthesized at different rates for different molecular species. Other information, such as chain elongation and desaturation, might also be derived through the use of this method.