On the dynamic order of structured Escherichia coli growth models

Reliable dynamic descriptions of cellular growth are important for many practical applications including bioreactor design and control. A chemically structured growth model of Escherichia coli has been formulated and herein we focus on finding the essential dynamic order of the metabolic part of this model. Standard linear analysis is applied and the main finding is that the model contains three essential modes of motion over the time scale of growth. The doubling time is successfully predicted from an unstable growth motion and the metabolite composition of the three modes of motion suggests that only a three pool metabolic model is necessary. The three pools correspond to important groups of macromolecules; protein, nucleic acids and cell wall constituents.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/37893/1/260290623_ftp.pd

Factors affecting plasmid production in Escherichia coli from a resource allocation standpoint

<p>Abstract</p> <p>Background</p> <p>Plasmids are being reconsidered as viable vector alternatives to viruses for gene therapies and vaccines because they are safer, non-toxic, and simpler to produce. Accordingly, there has been renewed interest in the production of plasmid DNA itself as the therapeutic end-product of a bioprocess. Improvement to the best current yields and productivities of such emerging processes would help ensure economic feasibility on the industrial scale. Our goal, therefore, was to develop a stoichiometric model of <it>Escherichia coli </it>metabolism in order to (1) determine its maximum theoretical plasmid-producing capacity, and to (2) identify factors that significantly impact plasmid production.</p> <p>Results</p> <p>Such a model was developed for the production of a high copy plasmid under conditions of batch aerobic growth on glucose minimal medium. The objective of the model was to maximize plasmid production. By employing certain constraints and examining the resulting flux distributions, several factors were determined that significantly impact plasmid yield. Acetate production and constitutive expression of the plasmid's antibiotic resistance marker exert negative effects, while low pyruvate kinase (Pyk) flux and the generation of NADPH by transhydrogenase activity offer positive effects. The highest theoretical yield (592 mg/g) resulted under conditions of no marker or acetate production, nil Pyk flux, and the maximum allowable transhydrogenase activity. For comparison, when these four fluxes were constrained to wild-type values, yields on the order of tens of mg/g resulted, which are on par with the best experimental yields reported to date.</p> <p>Conclusion</p> <p>These results suggest that specific plasmid yields can theoretically reach 12 times their current experimental maximum (51 mg/g). Moreover, they imply that abolishing Pyk activity and/or transhydrogenase up-regulation would be useful strategies to implement when designing host strains for plasmid production; mutations that reduce acetate production would also be advantageous. The results further suggest that using some other means for plasmid selection than antibiotic resistance, or at least weakening the marker's expression, would be beneficial because it would allow more precursor metabolites, energy, and reducing power to be put toward plasmid production. Thus far, the impact of eliminating Pyk activity has been explored experimentally, with significantly higher plasmid yields resulting.</p

Effect of plasmid replication deregulation via inc mutations on E. coli proteome & simple flux model analysis

When the replication of a plasmid based on sucrose selection is deregulated via the inc1 and inc2 mutations, high copy numbers (7,000 or greater) are attained while the growth rate on minimal medium is negligibly affected. Adaptions were assumed to be required in order to sustain the growth rate. Proteomics indicated that indeed a number of adaptations occurred that included increased expression of ribosomal proteins and 2-oxoglutarate dehydrogenase. The operating space prescribed by a basic flux model that maintained phenotypic traits (e.g. growth, byproducts, etc.) within typical bounds of resolution was consistent with the flux implications of the proteomic changes

Estudi sobre les hores més conflictives en un institut de secundària

Aquest Treball Final de Màster analitza la conflictivitat existent a les aules d'un institut de secundària. S'ha fet un estudi, amb dades reals de dos centres, sobre les incidències produïdes segons tres paràmetres que són la franja horària o hora lectiva, el curs i la matèria. Aquestes dades s'han representat i analitzat mitjançant taules i gràfics per poder fer una comparació. Els resultats obtinguts en els dos centres són sorprenentment coincidents pel que fa a conflictivitat per cursos i franges horàries, que es concentra al primer cicle i a la segona franja horària. Les diferències són una mica més grans pel que fa referència a les assignatures. Un cop identificada la franja horària més conflictiva del dia, s'ha dissenyat una proposta d'aplicació a l'aula relacionada amb tècniques de relaxació. Per desenvolupar la proposta s'ha fet un estudi de les tècniques existents a l'actualitat

Escherichia coil growth dynamics: A three-pool biochemically based description

A three-pool growth model of an individual Escherichia coli cell is described herein. The model is based on a previously developed chemically structured complex single cell growth model. The reduction in model complexity and the identification of the essential modes of motion, over the time scale of growth, is achieved by temporal decomposition and analysis of hierarchy in relaxation times. The three-pool model faithfully simulates the changes in cell size, cell shape, cell macromolecular composition, DNA initiation and termination periods, and the dependence of cell growth under abiotic glucose limitation. The predictions made by the reduced model compare favorably with both the experimental data and those of the full single cell model (SCM) without any parameter adjustments. The three-pool model has very few significant parameters and has the potential to find immediate practical use in bioreactor design and process control strategies. The model development illustrates the use of modal analysis to yield reduced physiologically realistic dynamic model of complex microbial system such as E. coll.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/37894/1/260310203_ftp.pd

Computational Design of Auxotrophy-Dependent Microbial Biosensors for Combinatorial Metabolic Engineering Experiments

Combinatorial approaches in metabolic engineering work by generating genetic diversity in a microbial population followed by screening for strains with improved phenotypes. One of the most common goals in this field is the generation of a high rate chemical producing strain. A major hurdle with this approach is that many chemicals do not have easy to recognize attributes, making their screening expensive and time consuming. To address this problem, it was previously suggested to use microbial biosensors to facilitate the detection and quantification of chemicals of interest. Here, we present novel computational methods to: (i) rationally design microbial biosensors for chemicals of interest based on substrate auxotrophy that would enable their high-throughput screening; (ii) predict engineering strategies for coupling the synthesis of a chemical of interest with the production of a proxy metabolite for which high-throughput screening is possible via a designed bio-sensor. The biosensor design method is validated based on known genetic modifications in an array of E. coli strains auxotrophic to various amino-acids. Predicted chemical production rates achievable via the biosensor-based approach are shown to potentially improve upon those predicted by current rational strain design approaches. (A Matlab implementation of the biosensor design method is available via http://www.cs.technion.ac.il/~tomersh/tools)

A Coarse-Grained Biophysical Model of E. coli and Its Application to Perturbation of the rRNA Operon Copy Number

We propose a biophysical model of Escherichia coli that predicts growth rate and an effective cellular composition from an effective, coarse-grained representation of its genome. We assume that E. coli is in a state of balanced exponential steadystate growth, growing in a temporally and spatially constant environment, rich in resources. We apply this model to a series of past measurements, where the growth rate and rRNA-to-protein ratio have been measured for seven E. coli strains with an rRNA operon copy number ranging from one to seven (the wild-type copy number). These experiments show that growth rate markedly decreases for strains with fewer than six copies. Using the model, we were able to reproduce these measurements. We show that the model that best fits these data suggests that the volume fraction of macromolecules inside E. coli is not fixed when the rRNA operon copy number is varied. Moreover, the model predicts that increasing the copy number beyond seven results in a cytoplasm densely packed with ribosomes and proteins. Assuming that under such overcrowded conditions prolonged diffusion times tend to weaken binding affinities, the model predicts that growth rate will not increase substantially beyond the wild-type growth rate, as indicated by other experiments. Our model therefore suggests that changing the rRNA operon copy number of wild-type E. coli cells growing in a constant rich environment does not substantially increase their growth rate. Other observations regarding strains with an altered rRNA operon copy number, such as nucleoid compaction and the rRNA operon feedback response, appear to be qualitatively consistent with this model. In addition, we discuss possible design principles suggested by the model and propose further experiments to test its validity