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

Major Role of Microbes in Carbon Fluxes during Austral Winter in the Southern Drake Passage

Carbon cycling in Southern Ocean is a major issue in climate change, hence the need to understand the role of biota in the regulation of carbon fixation and cycling. Southern Ocean is a heterogeneous system, characterized by a strong seasonality, due to long dark winter. Yet, currently little is known about biogeochemical dynamics during this season, particularly in the deeper part of the ocean. We studied bacterial communities and processes in summer and winter cruises in the southern Drake Passage. Here we show that in winter, when the primary production is greatly reduced, Bacteria and Archaea become the major producers of biogenic particles, at the expense of dissolved organic carbon drawdown. Heterotrophic production and chemoautotrophic CO2 fixation rates were substantial, also in deep water, and bacterial populations were controlled by protists and viruses. A dynamic food web is also consistent with the observed temporal and spatial variations in archaeal and bacterial communities that might exploit various niches. Thus, Southern Ocean microbial loop may substantially maintain a wintertime food web and system respiration at the expense of summer produced DOC as well as regenerate nutrients and iron. Our findings have important implications for Southern Ocean ecosystem functioning and carbon cycle and its manipulation by iron enrichment to achieve net sequestration of atmospheric CO2

Bowel management for the treatment of pediatric fecal incontinence

Fecal incontinence is a devastating underestimated problem, affecting a large number of individuals all over the world. Most of the available literature relates to the management of adults. The treatments proposed are not uniformly successful and have little application in the pediatric population. This paper presents the experience of 30 years, implementing a bowel management program, for the treatment of fecal incontinence in over 700 pediatric patients, with a success rate of 95%. The main characteristics of the program include the identification of the characteristics of the colon of each patient; finding the specific type of enema that will clean that colon and the radiological monitoring of the process

Morel-Lavallée lesion: A closed degloving injury that requires real attention

Morel-Lavallée lesions are post-traumatic, closed degloving injuries occurring deep to subcutaneous plane due to disruption of capillaries resulting in an effusion containing hemolymph and necrotic fat. Magnetic resonance imaging (MRI) is the modality of choice in the evaluation of Morel-Lavallée lesion. Early diagnosis and management is essential as any delay in diagnosis or missed lesion will lead to the effusion becoming infected or leading to extensive skin necrosis