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