Expanded flux variability analysis on metabolic network of Escherichia coli

Flux balance analysis, based on the mass conservation law in a cellular organism, has been extensively employed to study the interplay between structures and functions of cellular metabolic networks. Consequently, the phenotypes of the metabolism can be well elucidated. In this paper, we introduce the Expanded Flux Variability Analysis (EFVA) to characterize the intrinsic nature of metabolic reactions, such as flexibility, modularity and essentiality, by exploring the trend of the range, the maximum and the...     

The large-scale organization of metabolic networks

In a cell or microorganism, the processes that generate mass, energy, information transfer and cell-fate specification are seamlessly integrated through a complex network of cellular constituents and reactions. However, despite the key role of these networks in sustaining cellular functions, their large-scale structure is essentially unknown. Here we present a systematic comparative mathematical analysis of the metabolic networks of 43 organisms representing all three domains of life. We show that, despite significant variation in their individual constituents and pathways, these metabolic networks have the same topological scaling properties and show striking similarities to the inherent organization of complex non-biological systems. This may indicate that metabolic organization is not only identical for all living organisms, but also complies with the design principles of robust and error-tolerant scale-free networks, and may represent a common blueprint for the large-scale organization of interactions among all cellular constituents.     

Escherichia coli dam—dcm—菌株的研究进展

E.coli dam-dcm是一种在分子生物学技术中被广泛应用的菌株之一。Dam和Dcm是两种甲基转移酶,Dam识别GATC位点而Dcm识别CCA（T）GG位点[1],在E.coli细胞的生命活动中,dam基因产物在DNA的错配修复中具有重要作用,另外,dam的甲基化作用和DNA的复制和基因表达的调节等细胞生命活动过程,Dcm甲基化的生物学功能在很短的补丁修复有很重要的作用。Streptomyces(链霉菌）,Bacillus(芽胞杆菌）和Paracoccus（副球菌）的转化通过用dam和dcm位点没有甲基化的DNA可以得到很大的改善[6]。因为5－甲基胞嘧啶对肼有抗生,所以 从dcm缺陷的菌株分离到的DNA用Mazam和Gilbert法进行测序,结果更好[10]。     

大肠杆菌NADP特异性谷氨酸脱氢酶基因的克隆及其在大肠杆菌中的高效表达与纯化

谷氨酸脱氢酶是生物体内最主要的氧化脱氨基酶类，为了进一步研究其功能，我们以大肠杆菌DH5α菌株基因组DNA为模板和相应的寡聚脱氧核苷酸为引物，进行PCR扩增大肠杆菌NADP特异性谷氨酸脱氢酶基因(NADP-specific glutamate dehydrogenase，gdhA gene)，将所得DNA片断连接到质粒pUC18上，转化大肠杆菌DH5α，进行蓝白筛选和酶切鉴定，经测序证明序列正确无误后将gdhA基因连接到表达载体pTrcHisC上，在大肠杆菌BL21(DE3)中表达，经SDS-PAGE和双波长扫描分析，确定大肠杆菌谷氨酸脱氢酶在大肠杆菌中表达时以包涵体的形式存在，未能检测到可溶性蛋白的表达，表达量可达菌体总蛋白的15％以上．     

Global metabolic impacts of recent climate warming

Documented shifts in geographical ranges, seasonal phenology, community interactions, genetics and extinctions have been attributed to recent global warming. Many such biotic shifts have been detected at mid- to high latitudes in the Northern Hemisphere-a latitudinal pattern that is expected because warming is fastest in these regions. In contrast, shifts in tropical regions are expected to be less marked because warming is less pronounced there. However, biotic impacts of warming are mediated through physiology, and metabolic rate, which is a fundamental measure of physiological activity and ecological impact, increases exponentially rather than linearly with temperature in ectotherms. Therefore, tropical ectotherms (with warm baseline temperatures) should experience larger absolute shifts in metabolic rate than the magnitude of tropical temperature change itself would suggest, but the impact of climate warming on metabolic rate has never been quantified on a global scale. Here we show that estimated changes in terrestrial metabolic rates in the tropics are large, are equivalent in magnitude to those in the north temperate-zone regions, and are in fact far greater than those in the Arctic, even though tropical temperature change has been relatively small. Because of temperature's nonlinear effects on metabolism, tropical organisms, which constitute much of Earth's biodiversity, should be profoundly affected by recent and projected climate warming.     

Mutations in the active site of Escherichia coli phosphofructokinase

The enzyme-catalysed transfer of a phosphoryl group from ATP is an important reaction in a wide variety of biological processes. We demonstrate here the essential function of an aspartate group in the catalysis of phosphoryl transfer by Escherichia coli phosphofructokinase, and the minor role of an arginine residue. We have used oligonucleotide-directed mutagenesis to replace two amino-acid residues which X-ray analysis has shown to be close to the transferred phosphoryl group and we have analysed the forward and back reactions of the mutant enzymes by steady-state kinetics. Changing Asp 127 to Ser reduced the turnover number by a factor of 18,000 in the forward direction and 3,100 in the back reaction, and the Michaelis constant for fructose 1,6-bisphosphate in the reverse reaction by a factor of 45. This shows that this aspartate is a key residue in the rate enhancement by the enzyme, probably acting as a base in the reaction mechanism, and that it also destabilizes the product complex. Changing Arg 171 to Ser reduced the turnover numbers by about 3.4, showing that this arginine has only a minor effect on the catalysis.     

Escherichia coli swim on the right-hand side

The motion of peritrichously flagellated bacteria close to surfaces is relevant to understanding the early stages of biofilm formation and of pathogenic infection. This motion differs from the random-walk trajectories of cells in free solution. Individual Escherichia coli cells swim in clockwise, circular trajectories near planar glass surfaces. On a semi-solid agar substrate, cells differentiate into an elongated, hyperflagellated phenotype and migrate cooperatively over the surface, a phenomenon called swarming. We have developed a technique for observing isolated E. coli swarmer cells moving on an agar substrate and confined in shallow, oxidized poly(dimethylsiloxane) (PDMS) microchannels. Here we show that cells in these microchannels preferentially 'drive on the right', swimming preferentially along the right wall of the microchannel (viewed from behind the moving cell, with the agar on the bottom). We propose that when cells are confined between two interfaces--one an agar gel and the second PDMS--they swim closer to the agar surface than to the PDMS surface (and for much longer periods of time), leading to the preferential movement on the right of the microchannel. Thus, the choice of materials guides the motion of cells in microchannels.     

Distributed implementation of the genetic double-branch structure in Escherichia coli

Because of the simplicity of cells, the key to building biological computing systems may lie in constructing distributed systems based on cell-cell communication. Guided by a mathematical model, in this study we designed, simulated, and constructed a genetic double-branch structure in the bacterium Escherichia coli. This genetic double-branch structure is composed of a control cell and two reporter cells. The control cell can activate different reporter cells according to the input. Two quorum-sensing signal molecules, 3OC12- HSL and C4-HSL, form the wires between the control cell and the reporter cells. This study is a step toward scalable biological computation, and it may have many potential applications in biocomputing, biosensing, and biotherapy.     

有机磷水解酶的大肠杆菌细胞表面展示

PCR扩增来自黄杆菌ATCC27551的有机磷水解酶基因opd,直接与XcmⅠ酶切去除Ampr基因片段的pZXL-T连接,将opd克隆于锚定单元Lpp-OmpA编码序列下游,转化E.coliBL21(DE3).经抗性筛选、PCR鉴定和测序证实,成功构建了具有全细胞催化效应的大肠杆菌细胞表面展示工程菌.SDS-PAGE结果表明,工程菌能表达产生51 kD的融合蛋白.细胞表面展示的有机磷水解酶具有较高全细胞酶活性,用蛋白酶K消化处理重组菌表面蛋白可使其全细胞酶活降低90%.     

大肠杆菌卷曲菌毛及其致病性

大肠杆菌卷曲菌毛是其菌体表面的一种含纤维素样蛋白质附着器官,出现在大肠杆菌生理和病理过程中,可以通过黏附等作用介导大肠杆菌侵袭宿主.作为一种细菌淀粉样蛋白,卷曲菌毛有可能引起淀粉样蛋白相关疾病;卷曲菌毛可以诱导宿主炎症因子水平升高,引起脓毒血症;卷曲菌毛可以和纤维素等一起构成菌外基质,参与生物膜的形成.基于此,综述了大...