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

E. coli separatome-based protein expression and purification platform

Provided is a separatome-based peptide, polypeptide, and protein expression and purification platform based on the juxtaposition of the binding properties of host cell genomic peptides, polypeptides, and proteins with the characteristics and location of the corresponding genes on the host cell chromosome of E. coli. The separatome-based protein expression and purification platform quantitatively describes and identifies priority deletions, modifications, or inhibitions of certain gene products to increase chromatographic separation efficiency, defined as an increase in column capacity, column selectivity, or both, with emphasis on the former. Moreover, the separatome-based protein expression and purification platform provides a computerized knowledge tool that, given separatome data, and a target recombinant peptide, polypeptide, or protein, intuitively suggests strategies facilitating efficient product purification. The separatome-based protein expression and purification platform is an efficient bioseparation system that intertwines host cell expression systems and chromatography

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

Separatome-based protein expression and purification platform

Provided is a separatome-based recombinant peptide, polypeptide, and protein expression and purification platform based on the juxtaposition of the binding properties of host cell genomic peptides, polypeptides, and proteins with the characteristics and location of the corresponding genes on the host cell chromosome, such as that of E. coli, yeast, Bacillus subtilis or other prokaryotes, insect cells, mammalian cells, etc. This platform quantitatively describes and identifies priority deletions, modifications, or inhibitions of certain gene products to increase chromatographic separation efficiency, defined as an increase in column capacity, column selectivity, or both, with emphasis on the former. Moreover, the platform provides a computerized knowledge tool that, given separatome data and a target recombinant peptide, polypeptide, or protein, intuitively suggests strategies leading to efficient product purification. The separatome-based protein expression and purification platform is an efficient bioseparation system that intertwines host cell expression systems and chromatography

Growth-Rate Dependence Reveals Design Principles of Plasmid Copy Number Control

Genetic circuits in bacteria are intimately coupled to the cellular growth rate as many parameters of gene expression are growth-rate dependent. Growth-rate dependence can be particularly pronounced for genes on plasmids; therefore the native regulatory systems of a plasmid such as its replication control system are characterized by growth-rate dependent parameters and regulator concentrations. This natural growth-rate dependent variation of regulator concentrations can be used for a quantitative analysis of the design of such regulatory systems. Here we analyze the growth-rate dependence of parameters of the copy number control system of ColE1-type plasmids in E. coli. This analysis allows us to infer the form of the control function and suggests that the Rom protein increases the sensitivity of control

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

Investigation of Electromagnetic Wave Propagation Across Achiral-Chiral Interfaces and Fabry-Perot Type Slab Resonators Including Material Dispersion and Dielectric Loss

In this dissertation, Fresnel coefficients are examined for electromagnetic (EM) propagation across an achiral/chiral (ACC) boundary, and thereafter extended to cases involving slab-type resonator structures. An important factor to be noted is that while the numerical results presented in this research are accurate to the limits of our assumptions, one feature of a chiral material not taken into consideration in the early stages of work is the presence of dielectric losses. It turns out that a chiral (meta) material is usually also lossy via a complex dielectric permittivity. In the research presented in several chapters of this dissertation, the chiral dielectric has been assumed to be lossless, whereby all three material parameters (permittivity, permeability and chirality) are assumed to be real. Towards the latter chapters, dielectric losses are taken into account to make the results more compatible with practical cases. The results presented are aimed at observing the effects of loss on both the propagated fields themselves as decaying vectors, as well as on the amplitude and phase characteristics of the relevant Fresnel coefficients (amplitude as well as power or intensity). As is well known, upon both transmission and reflection from a chiral interface, the incident plane (specifically perpendicularly or s-polarized) wave transforms into two modes (right circular polarization (RCP) and left circular polarization (LCP)), propagating non-collinearly and collinearly for the transmitted and reflected modes respectively. This work focuses on certain anomalous properties pertinent to the chirality itself which provide novel insights into chiral materials. The dissertation highlights anomalies relative to the emergence of Brewster effects, tunability of Brewster angles and critical angles via the (dimensionless) chirality coefficient (? ?) over specific bands, total internal reflection (TIR), non-complementarity, mode evanescence, and also possible effective negative-index-like behavior in the chiral material. Extending the problem to include dispersive chiral dielectrics and discrete components may lead to potential applications such as tunable superlenses (with imaging and vision implications) and also devices including chiral Fabry-Perot etalons. Specific test cases for the propagation of a pulse-like waveform, as well a 2-D transparency with color and grey-scale variations have also been analyzed using appropriate dispersive models and Fresnel coefficients by use of MATLAB simulations. Extension to the cases involving two planar interfaces (ACC in the front and chiral/achiral (CAC) in the back), leading to entirely different optical characteristics compared with the standard achiral, lossless Fabry-Perot resonator. Results are first derived for the corresponding FCs in the case of a (CAC) propagation environment. Continuing with the lossless assumption, amplitude and intensity reflection and transmission characteristics of a chiral slab have been investigated in some detail. It is found that under chirality, there emerge singularities along the incident angle scale which in the limit exhibit total power coefficients exceeding 1 or drop below 1, which within an assumed ±10% variation are considered acceptable within computational errors, while when exceeding the assumed bound are interpreted as non-physical. The special cases of a chiral slab resonator in the thin film limit are also examined in some detail. The thin-film resonator is configured in both a uniform background and also a lithographic structure with three layers. Overall, chiral slabs (in those cases where energy conservation is satisfied) generally exhibit non-uniform transverse modes, with mode densities dependent on the slab thickness, chirality and also the permittivities of the layers. When dielectric loss is introduced in the chiral layer, the physics and related mathematical analyses of the problem become more complicated, with special attention being required to track the effect of a complex dielectric permittivity in the chiral region on the complex phasor fields, the boundary conditions applied at z = 0, and emergent Snell\u27s laws, and the interpretations of decaying fields in the chiral region and beyond, both in terms of their amplitude and phase behavior, as well as their intensity and energy conservation implications. Some results in this regard are presented with the understanding that further investigations with lossy chiral materials are currently ongoing

New strategies for designing inexpensive but selective bioadsorbants for environmental pollutants: Selection of specific ligands and their cell surface expression. 1998 annual progress report

'The broad, long term objective of the research plan is to develop exquisitely selective polypeptide metal chelators for the remediation of aqueous systems. A variety of polypeptide chelators will be developed and optimized ranging from antibodies to small peptides. Then, through unique molecular engineering approaches developed in the laboratories, the polypeptide chelators will be anchored directly on the surface of the cells that produce them. Thus, instead of using isolated biomolecules the authors will employ inexpensive genetically engineered whole cell adsorbents. Following a simple, easily scaleable treatment, the engineered cells can be used to manufacture an inexpensive, particulate adsorbent for metal removal. The authors are currently in year two of a three year program. Work has been focused on preparing the molecular biology constructs needed to carry out the optimization of a metal complex binding antibody, and on the isolation of a metal binding peptide.