Utilizing high-throughput experimentation to enhance specific productivity of an E.coli T7 expression system by phosphate limitation

<p>Abstract</p> <p>Background</p> <p>The specific productivity of cultivation processes can be optimized, amongst others, by using genetic engineering of strains, choice of suitable host/vector systems or process optimization (e.g. choosing the right induction time). A further possibility is to reduce biomass buildup in favor of an enhanced product formation, e.g. by limiting secondary substrates in the medium, such as phosphate. However, with conventional techniques (e.g. small scale cultivations in shake flasks), it is very tedious to establish optimal conditions for cell growth and protein expression, as the start of protein expression (induction time) and the degree of phosphate limitation have to be determined in numerous concerted, manually conducted experiments.</p> <p>Results</p> <p>We investigated the effect of different induction times and a concurrent phosphate limitation on the specific productivity of the T7 expression system <it>E.coli </it>BL21(DE3) pRhotHi-2-EcFbFP, which produces the model fluorescence protein EcFbFP upon induction. Therefore, specific online-monitoring tools for small scale cultivations (RAMOS, BioLector) as well as a novel cultivation platform (Robo-Lector) were used for rapid process optimization. The RAMOS system monitored the oxygen transfer rate in shake flasks, whereas the BioLector device allowed to monitor microbial growth and the production of EcFbFP in microtiter plates. The Robo-Lector is a combination of a BioLector and a pipetting robot and can conduct high-throughput experiments fully automated. By using these tools, it was possible to determine the optimal induction time and to increase the specific productivity for EcFbFP from 22% (for unlimited conditions) to 31% of total protein content of the <it>E.coli </it>cells via a phosphate limitation.</p> <p>Conclusions</p> <p>The results revealed that a phosphate limitation at the right induction time was suitable to redirect the available cellular resources during cultivation to protein expression rather than in biomass production. To our knowledge, such an effect was shown for the first time for an IPTG-inducible expression system. Finally, this finding and the utilization of the introduced high-throughput experimentation approach could help to find new targets to further enhance the production capacity of recombinant <it>E.coli</it>-strains.</p

Provisioning systems for a good life within planetary boundaries

The concept of provisioning systems has recently emerged as a promising way to understand the differences between levels of resource use and social outcomes observed across societies. However, the characteristics of provisioning systems remain poorly understood. Here, we make a new contribution to conceptualising provisioning systems and to understanding differences in the resource efficiency with which they achieve social outcomes. We define a provisioning system as a set of related elements that work together in the transformation of resources to satisfy a foreseen human need. We analyse six theories in terms of their contribution to understanding provisioning systems within the biophysical and social constraints of Raworth’s “Safe and Just Space” framework. We find that most of these theories fail to prioritise human needs and well-being, and do not incorporate explicit environmental limits. However, they provide important insights that we draw upon to identify six important provisioning system elements (households, markets, the commons, the state, techniques, and material stocks). Based on the theories, we also identify two important relationships between elements, namely feedbacks and power relations. We further propose the concept of “appropriating systems” as a component of provisioning systems. Appropriating systems reduce the resource efficiency of human well-being via rent extraction, and act as a barrier to meeting human needs at a sustainable level of resource use. We combine these concepts into a new framework, and discuss applications to energy systems

St. John's Daily Star, 1918-12-28

The St. John's Daily Star was published daily except Sunday between 17 April 1915 - 23 July 1921

Correlation between mass transfer coefficient kLa and relevant operating parameters in cylindrical disposable shaken bioreactors on a bench-to-pilot scale

Background: Among disposable bioreactor systems, cylindrical orbitally shaken bioreactors show important advantages. They provide a well-defined hydrodynamic flow combined with excellent mixing and oxygen transfer for mammalian and plant cell cultivations. Since there is no known universal correlation between the volumetric mass transfer coefficient for oxygen kLa and relevant operating parameters in such bioreactor systems, the aim of this current study is to experimentally determine a universal kLa correlation.Results: A Respiration Activity Monitoring System (RAMOS) was used to measure kLa values in cylindrical disposable shaken bioreactors and Buckingham's -Theorem was applied to define a dimensionless equation for kLa. In this way, a scale- and volume-independent kLa correlation was developed and validated in bioreactors with volumes from 2 L to 200 L. The final correlation was used to calculate cultivation parameters at different scales to allow a sufficient oxyge n supply of tobacco BY-2 cell suspension cultures.Conclusion: The resulting equation can be universally applied to calculate the mass transfer coefficient for any of seven relevant cultivation parameters such as the reactor diameter, the shaking frequency, the filling volume, the viscosity, the oxygen diffusion coefficient, the gravitational acceleration or the shaking diameter within an accuracy range of +/- 30%. To our knowledge, this is the first kLa correlation that has been defined and validated for the cited bioreactor system on a bench-to-pilot scale